Recombinant toxin fragments

ABSTRACT

Antigenic compositions are provided comprising a single chain polypeptide comprising first and second domains, wherein said first domain is a clostridial neurotoxin light chain or a fragment or a variant thereof and is capable of cleaving one or more vesicle or plasma membrane associated proteins essential to exocytosis; and said second domain is a clostridial neurotoxin heavy chain H N  portion or a fragment or a variant thereof, wherein said second domain is capable of (i) translocating the polypeptide into a cell or (ii) increasing the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocating the polypeptide into a cell and increasing the solubility of the polypeptide compared to the solubility of the first domain on its own; and wherein the second domain lacks a functional C-terminal part of a clostridial neurotoxin heavy chain designated H C  thereby rendering the polypeptide incapable of binding to cell surface receptors that are the natural cell surface receptors to which native clostridial neurotoxin binds. Antibodies that bind to the polypeptides, and compositions comprising these antibodies, are also provided, as are DNA vaccines comprising polynucleotides that encode these polypeptides. 
     The antigenic and antibody compositions, and the DNA vaccine compositions, can be used in methods of immunising against, or treating, clostridial neurotoxin poisoning in a subject by administering to that subject a therapeutically effective amount of the composition.

This application is a continuation-in-part of U.S. application Ser. No.10/241,596 filed on Sep. 12, 2002, now U.S. Pat. No. 7,192,596, which isa continuation-in-part of U.S. application Ser. No. 09/255,829, filedFeb. 23, 1999, now U.S. Pat. No. 6,461,617, which is a continuation ofInternational Application No. PCT/GB97/02273, filed Aug. 22, 1997, nowinactive, which is a continuation-in-part of U.S. application Ser. No.08/782,893, filed Dec. 27, 1996, now abandoned.

This invention relates to recombinant toxin fragments, to DNA encodingthese fragments and to their uses such as in a vaccine and for in vitroand in vivo purposes.

The clostridial neurotoxins are potent inhibitors of calcium-dependentneurotransmitter secretion in neuronal cells. They are currentlyconsidered to mediate this activity through a specific endoproteolyticcleavage of at least one of three vesicle or pre-synaptic membraneassociated proteins VAMP, syntaxin or SNAP-25 which are central to thevesicle docking and membrane fusion events of neurotransmittersecretion. The neuronal cell targeting of tetanus and botulinumneurotoxins is considered to be a receptor mediated event followingwhich the toxins become internalised and subsequently traffic to theappropriate intracellular compartment where they effect theirendopeptidase activity.

The clostridial neurotoxins share a common architecture of a catalyticL-chain (LC, ca 50 kDa) disulphide linked to a receptor binding andtranslocating H-chain (HC, ca 100 kDa). The HC polypeptide is consideredto comprise all or part of two distinct functional domains. Thecarboxy-terminal half of the HC (ca 50 kDa), termed the H_(C) domain, isinvolved in the high affinity, neurospecific binding of the neurotoxinto cell surface receptors on the target neuron, whilst theamino-terminal half, termed the H_(N) domain (ca 50 kDa), is consideredto mediate the translocation of at least some portion of the neurotoxinacross cellular membranes such that the functional activity of the LC isexpressed within the target cell. The H_(N) domain also has theproperty, under conditions of low pH, of forming ion-permeable channelsin lipid membranes, this may in some manner relate to its translocationfunction.

For botulinum neurotoxin type A (BoNT/A) these domains are considered toreside within amino acid residues 872-1296 for the H_(C), amino acidresidues 449-871 for the H_(N) and residues 1-448 for the LC. Digestionwith trypsin effectively degrades the H_(C) domain of the BoNT/A togenerate a non-toxic fragment designated LH_(N), which is no longer ableto bind to and enter neurons (FIG. 1). The LH_(N) fragment so producedalso has the property of enhanced solubility compared to both the parentholotoxin and the isolated LC.

It is therefore possible to provide functional definitions of thedomains within the neurotoxin molecule, as follows:

-   (A) clostridial neurotoxin light chain:    -   a metalloprotease exhibiting high substrate specificity for        vesicle and/or plasma-membrane associated proteins involved in        the exocytotic process. In particular, it cleaves one or more of        SNAP-25, VAMP (synaptobrevin/cellubrevin) and syntaxin.-   (B) clostridial neurotoxin heavy chain H_(N) domain:    -   a portion of the heavy chain which enables translocation of that        portion of the neurotoxin molecule such that a functional        expression of light chain activity occurs within a target cell.    -   the domain responsible for translocation of the endopeptidase        activity, following binding of neurotoxin to its specific cell        surface receptor via the binding domain, into the target cell.    -   the domain responsible for formation of ion-permeable pores in        lipid membranes under conditions of low pH.    -   the domain responsible for increasing the solubility of the        entire polypeptide compared to the solubility of light chain        alone.-   (C) clostridial neurotoxin heavy chain H_(C) domain.    -   a portion of the heavy chain which is responsible for binding of        the native holotoxin to cell surface receptor(s) involved in the        intoxicating action of clostridial toxin prior to        internalisation of the toxin into the cell.

The identity of the cellular recognition markers for these toxins iscurrently not understood and no specific receptor species have yet beenidentified although Kozaki et al. (1996) Neurosci. Lett., 208(2), pp.105-8, have reported that synaptotagmin may be the receptor forbotulinum neurotoxin type B. It is probable that each of the neurotoxinshas a different receptor.

It is desirable to have positive controls for toxin assays, to developclostridial toxin vaccines and to develop therapeutic agentsincorporating desirable properties of clostridial toxin.

However, due to its extreme toxicity, the handling of native toxin ishazardous.

The present invention seeks to overcome or at least ameliorate problemsassociated with production and handling of clostridial toxin.

Accordingly, the invention provides a polypeptide comprising first andsecond domains, wherein said first domain is adapted to cleave one ormore vesicle or plasma-membrane associated proteins essential toneuronal exocytosis and wherein said second domain is adapted (i) totranslocate the polypeptide into the cell or (ii) to increase thesolubility of the polypeptide compared to the solubility of the firstdomain on its own or (iii) both to translocate the polypeptide into thecell and to increase the solubility of the polypeptide compared to thesolubility of the first domain on its own, said polypeptide being freeof clostridial neurotoxin and free of any clostridial neurotoxinprecursor that can be converted into toxin by proteolytic action.Accordingly, the invention may thus provide a single polypeptide chaincontaining a domain equivalent to a clostridial toxin light chain and adomain providing the functional aspects of the H_(N) of a clostridialtoxin heavy chain, whilst lacking the functional aspects of aclostridial toxin H_(C) domain.

In a preferred embodiment, the present invention provides a single chainpolypeptide comprising first and second domains, wherein:

said first domain is a clostridial neurotoxin light chain or a fragmentor a variant thereof, wherein said first domain is capable of cleavingone or more vesicle or plasma membrane associated proteins essential toexocytosis; and

said second domain is a clostridial neurotoxin heavy chain H_(N) portionor a fragment or a variant thereof, wherein said second domain iscapable of (i) translocating the polypeptide into a cell or (ii)increasing the solubility of the polypeptide compared to the solubilityof the first domain on its own or (iii) both translocating thepolypeptide into a cell and increasing the solubility of the polypeptidecompared to the solubility of the first domain on its own; and whereinthe second domain lacks a functional C-terminal part of a clostridialneurotoxin heavy chain designated H_(C) thereby rendering thepolypeptide incapable of binding to cell surface receptors that are thenatural cell surface receptors to which native clostridial neurotoxinbinds.

In the above preferred embodiment, the first domain is qualified by arequirement for the presence of a particular cleavage function. Saidcleavage function may be present when the light chain (L-chain)component is part of the single chain polypeptide molecule per se.Alternatively, the cleavage function may be substantially latent in thesingle chain polypeptide molecule, and may be activated by proteolyticcleavage of the single polypeptide between the first and second domainsto form, for example, a dichain polypeptide molecule comprising thefirst and second domains disulphide bonded together.

The first domain is based on a clostridial neurotoxin light chain(L-chain), and embraces both fragments and variants of said L-chain solong as these components possess the requisite cleavage function. Anexample of a variant is an L-chain (or fragment thereof) in which one ormore amino acid residues has been altered vis-a-vis a native clostridialL-chain sequence. In one embodiment, the modification may involve one ormore conservative amino acid substitutions. Other modifications mayinclude the removal or addition of one or more amino acid residuesvis-a-vis a native clostridial L-chain sequence. However, any suchfragment or variant must retain the aforementioned cleavage function.

The structure of clostridial neurotoxins was well known prior to thepresent invention—see, for example, Kurazono et al (1992) J. Biol.Chem., 267, 21, pp. 14721-14729. In particular, the Kurazono paperdescribes the minimum Domains required for cleavage activity (eg.proteolytic enzyme activity) of a clostridial neurotoxin L-chain.Similar discussion is provided by Poulain et al (1989) Eur. J. Biochem.,185, pp. 197-203, by Zhou et al (1995), 34, pp. 15175-15181, and byBlaustein et al (1987), 226, No. 1, pp. 115-120.

By way of exemplification, Table II on page 14726 of Kurazono et al.(1992) illustrates a number of L-chain deletion mutants (bothamino-terminal and carboxy-terminal L-chain deletion mutants areillustrated). Such mutants, together with other L-chain mutantscontaining, for example, similar amino acid deletions or conservativeamino acid substitutions are embraced by the first domain definition ofthe present invention provided that the L-chain component in questionhas the requisite cleavage activity.

Prior to the present application a number of conventional, simple assayswere available to allow a skilled person to routinely confirm whether agiven L-chain (or equivalent L-chain component) had the requisitecleavage activity. These assays are based on the inherent ability of afunctional L-chain to effect peptide cleavage of specific vesicle orplasma membrane associated proteins (eg. synaptobrevin, syntaxin, orSNAP-25) involved in neuronal exocytosis, and simply test for thepresence of the cleaved product/s of said proteolytic reaction.

For example, in a rough-and-ready assay, SNAP-25 (or synaptobrevin, orsyntaxin) may be challenged with a test L-chain (or equivalent L-chaincomponent), and then analysed by SDS-PAGE peptide separation techniques.Subsequent detection of peptides (eg. by silver staining) havingmolecular weights corresponding to the cleaved products of SNAP-25 (orother component of the neurosecretory machinery) would indicate thepresence of an L-chain (or equivalent L-chain component) possessing therequisite cleavage activity.

In an alternative assay, SNAP-25 (or a different neuronal exocytosismolecule) may be challenged with a test L-chain (or equivalent L-chaincomponent), and the cleavage products subjected to antibody detection asdescribed in PCT/GB95/01279 (ie. WO95/33850) in the name of the presentApplicant, Microbiological Research Authority. In more detail, aspecific antibody is employed for detecting the cleavage of SNAP-25,which antibody recognises cleaved SNAP-25 but not uncleaved SNAP-25.Identification of the cleaved product by the antibody confirms thepresence of an L-chain (or equivalent L-chain component) possessing therequisite cleavage activity. By way of exemplification, such a method isdescribed in Examples 2 and 3 of PCT/GB96/00916 (ie. WO96/33273), alsoin the name of Microbiological Research Authority.

In a preferred embodiment of the present invention, the second domain isqualified by the ability to provide one or both of two functions, namely(i) translocation and/or (ii) increased solubility of the first domain.

The second domain is based on a H_(N) portion of a clostridialneurotoxin, which portion has been extensively described andcharacterised in the literature. Particular mention is made to Kurazonoet al (1992) in which the structure of clostridial neurotoxin heavychains is discussed together with the functions associated with theH_(N) and H_(C) portions thereof [see, for example, the bottomillustration in FIG. 1 on page 14722 of Kurazono et al (1992)]. In moredetail, the H_(N) domain is a domain of a clostridial neurotoxin thatfunctions to translocate a clostridial L-chain across the endosomalmembrane of a vesicle, and is synonymous with the H₂ domain of aclostridial neurotoxin [see the bottom left-hand column and footer onpage 197 of Poulain, B. et al (1989); see FIG. 1 in Blaustein, R. et al(1987); and see also the sentence bridging pages 178 and 179 of Shone,C. et al (1987), Eur. J. Biochem., 167, pp. 175-180].

The second domain definition of the present invention includes fragmentsand variants of the H_(N) portion of a clostridial neurotoxin so long asthese components provide the requisite (I) translocation and/or (ii)improved solubility function. An example of a variant is an H_(N)portion (or fragment thereof) in which one or more amino acid residueshas been altered vis-a-vis a native clostridial H_(N) domain sequence.In one embodiment, the modification may involve one or more conservativeamino acid substitutions. Other modifications may include the removal oraddition of one or more amino acid residues vis-a-vis a nativeclostridial H_(N) sequence. However, any such fragment or variant mustprovide the aforementioned (i) translocation and/or (ii) improvedsolubility function.

The (i) translocation and (ii) improved solubility functions are nowdescribed in more detail.

Prior to the present application a number of conventional, simple assayswere available to allow a skilled person to routinely confirm whether aparticular clostridial neurotoxin H_(N) portion (or equivalent H_(N)component) had the requisite translocation function. In this respect,particular mention is made to the assays described in Shone et al.(1987) and Blaustein et al. (1987), which are now discussed.

These papers describe studies of the translocation function ofclostridial neurotoxins, and demonstrate that the ability of saidneurotoxins to form channels is associated with the presence of atranslocation function.

Shone et al. (1987) describes an assay employing artificial liposomesloaded with potassium phosphate buffer (pH 7.2) and radiolabelled NAD.Thus, to confirm whether a test H_(N) portion (or equivalent H-chaincomponent) of a clostridial neurotoxin has the requisite translocationfunction, the artificial liposomes are challenged with the test H_(N)portion. The release of K⁺ and NAD from the liposomes is indicative of achannel-forming activity, and thus the presence of a translocationfunction.

An alternative assay is described by Blaustein et al. (1987), whereinplanar phospholipid bilayer membranes are used to test forchannel-forming activity. Salt solutions on either side of the membraneare buffered at different pH—on the cis side, pH 4.7 or 5.5 and on thetrans side, pH 7.4. Thus, to confirm whether a H_(N) portion (orequivalent H-chain component) of a clostridial neurotoxin has therequisite translocation function, the test H_(N) portion is added to thecis side of the membrane and electrical measurements made under voltageclamp conditions, in order to monitor the flow of current across themembrane (see paragraph 2.2 on pages 116-118). The presence of a desiredtranslocation activity is confirmed by a steady rate of channel turn-on(see paragraph 3 on page 118).

Turning now to the second heavy chain function, namely (ii) increasedsolubility of the first domain. A conventional problem associated withthe preparation of a clostridial neurotoxin L-chain molecules is thatsaid L-chain molecules generally possess poor solubilitycharacteristics. Thus, in one embodiment of the present invention, thefusion of a second domain (based on a H_(N) portion of a clostridialneurotoxin) to the L-chain increases the solubility of the L-chain.Similarly, the addition of a second domain to a L-chain equivalentmolecule (eg. a fragment, or variant of a L-chain) increases thesolubility of the L-chain equivalent molecule.

Prior to the present application a number of conventional, simple assayswere available to allow a skilled person to routinely confirm whether aparticular clostridial neurotoxin H_(N) portion (or equivalent H_(N)component) had the requisite ability to increase the solubility of aL-chain (or equivalent L-chain component). The most common method toassess solubility is through use of centrifugation, followed by a rangeof protein determination methods. For example, lysed E. coli cellscontaining expressed clostridial endopeptidase are centrifuged at25,000×g for 15 minutes to pellet cell debris and aggregated proteinmaterial. Following removal of the supernatant (containing solubleprotein) the cell debris can be reconstituted in SDS-containing samplebuffer (to solubilise the poorly soluble protein), prior to analysis ofthe two fractions by SDS-PAGE. Coomassie blue staining ofelectrophoresed protein, followed by densitometric analysis of therelevant protein band, facilitates a semi-quantitative analysis ofsolubility of expressed protein.

A further requirement of the single polypeptide molecule according to apreferred embodiment of the present invention is that the second domainlacks a functional C-terminal part of a clostridial neurotoxin heavychain designated H_(C), thereby rendering the polypeptide incapable ofbinding to cell surface receptors that are the natural cell surfacereceptors to which a native clostridial neurotoxin binds. Thisrequirement is now discussed in more detail, and reference to incapableof binding throughout the present specification is to be interpreted assubstantially incapable of binding, or reduced in binding ability whencompared with native clostridial neurotoxin.

It has been well documented, for example in the above-describedliterature and elsewhere, that native clostridial neurotoxin binds tospecific target cells through a binding interaction that involves theH_(C) domain of the toxin heavy chain and a specific receptor on thetarget cell.

However, in contrast to native neurotoxin, the single polypeptidemolecules according to a preferred embodiment of the. present inventionlack a functional H_(C) domain of native clostridial neurotoxin. Thus,the preferred single polypeptide molecules of the present invention arenot capable of binding to the specific receptors targeted by nativeclostridial neurotoxin.

Prior to the present application a number of conventional, simple assayswere available to allow a skilled person to routinely confirm whether aparticular clostridial neurotoxin H_(N) portion (or equivalent H_(N)component) lacked the binding ability of native clostridial neurotoxin.In this respect, particular mention is made to the assays described. byShone et al. (1985) Eur. J. Biochem., 151(1), pp. 75-82, and by Black &Dolly (1986) J. Cell. Biol., 103, pp. 521-534. The basic Shone et al(1985) method has been recently repeated in Sutton et al (2001), 493,pp. 45-49 to assess the binding ability of tetanus toxins.

These papers describe simple methods for assessing binding of theH-chain of a clostridial neurotoxin to its target cells, motor neurons.Hence, these methods provide a means for routinely determining whether amodification to the H-chain results in a loss of or reduced nativebinding affinity of the H-chain for motor neurons. The methods are nowdiscussed in more detail.

The Shone et al (1985) method is based on a competitive binding assay inwhich test neurotoxin H-chain fragments are compared with radiolabellednative neurotoxin in their ability to bind to purified ratcerebrocortical synaptosomes (ie. native toxin target cells).

A reduction of H_(C) function (ie. binding ability) is demonstrated by areduced ability of the test H-chain fragments to compete with thelabelled intact toxin for binding to the synaptosomes (see page 76,column 1 to line 51-column 2, line 5).

Sutton et al. (2001) carried out similar competitive binding experimentsusing radiolabelled intact tetanus neurotoxin (TeNT) and unlabelledsite-directed (TeNT) mutants. As above, a positive result in the assayis demonstrated by an inability of the mutant fragments to compete withthe labelled TeNT for binding to synaptosomes.

An alternative approach is described by Black & Dolly (1986), whichmethod employed electron microscopic autoradiography to visually assessbinding of radiolabelled clostridial neurotoxins at the vertebrateneuromuscular junction, both in vivo and in vitro. Thus, this assayrepresents a simple visual method for confirming whether a test H_(N)domain (or equivalent H_(N) component) lacks a functional H_(C) domain.

There are numerous ways by which a second domain that lacks a functionalH_(C) domain may be prepared. In this respect, inactivation of the H_(C)domain may be achieved at the amino acid level (eg. by use of aderivatising chemical, or a proteolytic enzyme), or at the nucleic acidlevel (eg. by use of site-directed mutagenesis, nucleotide/s insertionor deletion or modification, or by use of truncated nucleic acid).

For example, it would be routine for a skilled person to select aconventional derivatising chemical or proteolytic agent suitable forremoval or modification of the H_(c) domain. Standard derivatisingchemicals and proteolytic agents are readily available in the art, andit would be routine for a skilled person to confirm that saidchemicals/agents provide an H_(N) domain with reduced or removed nativebinding affinity by following any one of a number of simple tests suchas those described above.

Conventional derivatising chemicals may include any one of thefollowing, which form a non-exhaustive list of examples:

-   -   (1) tyrosine derivatising chemicals such as anhydrides, more        specifically maleic anhydride;    -   (2) diazonium based derivatising chemicals such as        bis-Diazotized o-Tolidine, and diazotized p-aminobenzoyl        biocytin;    -   (3) EDC (1-ethyl 1-3-(3-dimethylaminopropyl) carbodiimide        hydrochloride);    -   (4) isocyanate based derivatising chemicals such as dual        treatment with tetranitromethane followed by sodium dithionite;        and    -   (5) iodinating derivatising chemicals such as chloramine-T        (N-chlorotoluene sulfonamide) or IODO-GEN        (1,3,4,6-tetrachloro-3a,ba-diphenylglycouril).

Conventional proteolytic agents may include any one of the following,which form a non-exhaustive list of examples:

-   -   (1) trypsin [as demonstrated in Shone et al (1985)];    -   (2) proline endopeptidase    -   (3) lys C proteinase;    -   (4) chymotrypsin;    -   (5) thermolysin; and    -   (6) arg C proteinase.

Alternatively, conventional nucleic acid mutagenesis methods may beemployed to generate modified nucleic acid sequences that encode seconddomains lacking a functional H_(C) domain. For example, mutagenesismethods such as those described in Kurazono et al (1992) may beemployed. A range of systems for mutagenesis of DNA are available, basedon the DNA manipulation techniques described by: Kunkel T. (1985) Proc.Natl. Acad. Sci. USA, 82, pp. 488-492; Taylor, J. W. et al. (1985)Nucleic Acids Res. 13, pp. 8749-8764 (1995); and Deng G. & Nickeloff J.A. (1992) Anal. Biochem., 200, pp. 81-88.

According to all general aspects of the present invention, a polypeptideof the invention can be soluble but lack the translocation function of anative toxin-this is of use in providing an immunogen for vaccinating orassisting to vaccinate an individual against challenge by toxin. In aspecific embodiment of the invention described in an example below apolypeptide designated LH₄₂₃/A elicited neutralising antibodies againsttype A neurotoxin. A polypeptide of the invention can likewise thus berelatively insoluble but retain the translocation function of a nativetoxin—this is of use if solubility is imparted to a composition made upof that polypeptide and one or more other components by one or more ofsaid other components.

The first domain of the polypeptide of the invention cleaves one or morevesicle or plasma-membrane associated proteins essential to the specificcellular process of exocytosis, and cleavage of these proteins resultsin inhibition of exocytosis, typically in a non-cytotoxic manner. Thecell or cells affected are not restricted to a particular type orsubgroup but can include both neuronal and non-neuronal cells. Theactivity of clostridial neurotoxins in inhibiting exocytosis has,indeed, been observed almost universally in eukaryotic cells expressinga relevant cell surface receptor, including such diverse cells as fromAplysia (sea slug), Drosophila (fruit fly) and mammalian nerve cells,and the activity of the first domain is to be understood as including acorresponding range of cells.

The polypeptide of the invention may be obtained by expression of arecombinant nucleic acid, preferably a DNA, and is a single polypeptide,that is to say not cleaved into separate light and heavy chain domains.The polypeptide is thus available in convenient and large quantitiesusing recombinant techniques.

In a polypeptide according to the invention, said first domainpreferably comprises a clostridial toxin light chain or a fragment orvariant of a clostridial toxin light chain. The fragment is optionallyan N-terminal, or C-terminal fragment of the light chain, or is aninternal fragment, so long as it substantially retains the ability tocleave the vesicle or plasma-membrane associated protein essential toexocytosis. The minimal domains necessary for the activity of the lightchain of clostridial toxins are described in J. Biol. Chem., Vol. 267,No. 21, July 1992, pages 14721-14729. The variant has a differentpeptide sequence from the light chain or from the fragment, though ittoo is capable of cleaving the vesicle or plasma-membrane associatedprotein. It is conveniently obtained by insertion, deletion and/orsubstitution of a light chain or fragment thereof. In embodiments of theinvention described below a variant sequence comprises (i) an N-terminalextension to a clostridial toxin light chain or fragment (ii) aclostridial toxin light chain or fragment modified by alteration of atleast one amino acid (iii) a C-terminal extension to a clostridial toxinlight chain or fragment, or (iv) combinations of 2 or more of (i)-(iii).

The first domain preferably exhibits endopeptidase activity specific fora substrate selected from one or more of SNAP-25, synaptobrevin/VAMP andsyntaxin. The clostridial toxin is preferably botulinum toxin or tetanustoxin.

In one embodiment of the invention described in an example below, thetoxin light chain and the portion of the toxin heavy chain are ofbotulinum toxin type A. In a further embodiment of the inventiondescribed in an example below, the toxin light chain and the portion ofthe toxin heavy chain are of botulinum toxin type B. The polypeptideoptionally comprises a light chain or fragment or variant of one toxintype and a heavy chain or fragment or variant of another toxin type.

In a polypeptide according to the invention said second domainpreferably comprises a clostridial toxin heavy chain H_(N) portion or afragment or variant of a clostridial toxin heavy chain H_(N) portion.The fragment is optionally an N-terminal or C-terminal or internalfragment, so long as it retains the function of the H_(N) domain.Teachings of regions within the H_(N) responsible for its function areprovided for example in Biochemistry 1995, 34, pages 15175-15181 andEur. J. Biochem, 1989, 185, pages 197-203. The variant has a differentsequence from the H_(N) domain or fragment, though it too retains thefunction of the H_(N) domain. It is conveniently obtained by insertion,deletion and/or substitution of a H_(N) domain or fragment thereof. Inembodiments of the invention, described below, it comprises (i) anN-terminal extension to a H_(N) domain or fragment, (ii) a C-terminalextension to a H_(N) domain or fragment, (iii) a modification to a H_(N)domain or fragment by alteration of at least one amino acid, or (iv)combinations of 2 or more of (i)-(iii). The clostridial toxin ispreferably botulinum toxin or tetanus toxin.

The invention also provides a polypeptide comprising a clostridialneurotoxin light chain and a N-terminal fragment of a clostridialneurotoxin heavy chain, the fragment preferably comprising at least 423of the N-terminal amino acids of the heavy chain of botulinum toxin typeA, 417 of the N-terminal amino acids of the heavy chain of botulinumtoxin type B or the equivalent number of N-terminal amino acids of theheavy chain of other types of clostridial toxin such that the fragmentpossesses an equivalent alignment of homologous amino acid residues.

These polypeptides of the invention are thus not composed of two or morepolypeptides, linked for example by di-sulphide bridges into compositemolecules. Instead, these polypeptides are single chains and are notactive or their activity is significantly reduced in an in vitro assayof neurotoxin endopeptidase activity.

Further, the polypeptides may be susceptible to be converted into a formexhibiting endopeptidase activity by the action of a proteolytic agent,such as trypsin. In this way it is possible to control the endopeptidaseactivity of the toxin light chain.

In further embodiments of the invention, the polypeptide contains anamino acid sequence modified so that (a) there is no protease sensitiveregion between the LC and H_(N) components of the polypeptide, or (b)the protease sensitive region is specific for a particular protease.This latter embodiment is of use if it is desired to activate theendopeptidase activity of the light chain in a particular environment orcell. Though, in general, the polypeptides of the invention areactivated prior to administration.

More generally, a proteolytic cleavage site may be introduced betweenany two domains of the single chain polypeptide molecule.

For example, a cleavage site may be introduced between the first andsecond domains such that cleavage thereof converts the single chainpolypeptide molecule into a dichain polypeptide structure wherein thefirst and second domains are linked together by a disulphide bond.Specific Examples of such molecules are provided by SEQ IDs 11-18 of thepresent application in which an Factor Xa cleavage site has beenintroduced between the first domain (L-chain) and the second domain(H_(N)).

A range of peptide sequences having inherent cleavage sites areavailable for insertion into the junction between one or more domains ofa polypeptide according to the present invention. For example, insertionof a cleavage site between the first (L-chain) and second (H_(N))domains may result in a single polypeptide chain molecule that isproteolytically cleavable to form a dichain polypeptide in which thefirst and second domains are held together by a disulphide bond betweenthe first and second domains. The proteolytic cleavage may be performedin vitro prior to use, or in vivo by cell specific activation throughintracellular proteolytic action.

Alternatively (or additionally), a cleavage site may be introducedbetween the second and third domains, or between the purification tagand the polypeptide of the present invention. The third domain andpurification tag aspects of the present invention are discussed in moredetail below.

To facilitate convenient insertion of a range of cleavage sites into thejunction between the LC and H_(N) domains, it is preferable to preparean expression clone that can serve as a template for future clonedevelopment. Such a template is represented by SEQ ID 103, in which theDNA encoding LH_(N)/B has been modified by standard mutagenesistechniques to incorporate unique restriction enzyme sites. Toincorporate new cleavage sites at the junction requires simple insertionof novel oligonucleotides encoding the new cleavage site.

Suitable cleavage sites include, but are not limited to, those describedin Table 1.

TABLE 1 Cleavage site (eg. between the first and second domains forLH_(N) activation) Amino acid Protease sequence of recognition site SEQID exemplification Factor Xa I-E/D-G-R

71/72, 33/34, 55/56, 57/58, 115/116, 117/118, 119/120, 121/122Enterokinase D-D-D-D-K

69/70, 31/32, 29/30, 43/44, 45/46, 113/114, 111/112, 59/60, 61/62,63/64, 65/66, 79/80, 81/82, 83-98, 105/106, 107/108 PrecissionL-E-V-L-F-Q

 G-P 75/76, 35/36, 51/52, 53/54 Thrombin L-V-P-R

 G-S 77/78, 37/38, 47/48, 49/50, 99/100 Genenase H-Y

 or Y

-H TEV E-N-L-Y-F-Q

 G 101/102 Furin R-X-X-R

, preferred R-X-K/R-R

(wherein X = any amino acid)

In some cases, the use of certain cleavage sites and correspondingproteolytic enzymes (eg. precission, thrombin) will leave a shortN-terminal extension on the polypeptide at a position C-terminal to thecleavage site (see the

cleavage pattern for the exemplified proteases in Table 1).

Peptide sequences may be introduced between any two domains tofacilitate specific cleavage of the domains at a later stage. Thisapproach is commonly used in proprietary expression systems for cleavageand release of a purification tag (eg. maltose-binding protein (MBP),glutathione S-transferase (GST), polyhistidine tract (His6)) from afusion protein that includes the purification tag. In this respect, thepurification tag is preferably fused to the N- or C-terminus of thepolypeptide in question.

The choice of cleavage site may have a bearing on the precise nature ofthe N-terminus (or C-terminus) of the released polypeptide. Toillustrate this, identical LH_(N)/B fragments produced in suchproprietary systems are described in SEQ ID 88, 94, 96, 98, in which theN-terminal extensions to the LH_(N)/B sequence are ISEFGS, GS, SPGARGS &AMADIGS respectively. In the case of LH_(N)/C fragments, SEQ ID 126, 128& 130 describe the N-terminal sequences VPEFGSSRVDH, ISEFGSSRVDH andVPEFGSSRVDH following release of the LH_(N)/C fragment from its fusiontag by enterokinase, genenase and Factor Xa respectively. Each of theseextension peptide sequences is an example of a variant L-chain sequenceof the present invention. Similarly, if the purification tag were to befused to the C-terminal end of the second domain, the resulting cleavedpolypeptide (ie. fusion protein minus purification tag) would includeC-terminal extension amino acids. Each of these extension peptidesprovides an example of a variant H_(N) portion of the present invention.

In some cases, cleavage at a specific site, for example, between apurification tag and a polypeptide of the present invention may be oflower efficiency than desired. To address this potential problem, thepresent Applicant has modified proprietary vectors in two particularways, which modifications may be employed individually or in combinationwith each other. Whilst said modifications may be applied to cleavagesites between any two domains in a polypeptide or fusion proteinaccording to the present invention, the following discussion simplyillustrates a purification tag-first domain cleavage event.

First, the DNA is modified to include an additional peptide spacersequence, which optionally may represent one or more additional cleavagesites, at the junction of the purification tag and the polypeptide.Examples of the full-length expressed polypeptide from this approach arepresented in SEQ ID 86, 90 & 92. Such an approach has resulted inefficient cleavage and release of the polypeptide of interest. Dependingon the presence and nature of any intra-polypeptide cleavage sites (eg.between the first and second domains), cleavage of the purification tagfrom the fusion protein may occur simultaneously to proteolytic cleavagebetween the first and second domains. Alternatively, release of thepurification tag may occur without proteolytic cleavage between thefirst and second domains. These two cleavage schemes are illustrated inFIG. 14.

Depending on the cleavage enzyme chosen, this strategy may result in ashort amino acid extension to the N-terminus (or C-terminus) of thepolypeptide. For example, in the case of SEQ ID 92, cleavage of theexpressed product with enterokinase results in two polypeptides coupledby a single disulphide bond at the first domain-second domain junction(ie. the L chain-H_(N) junction), with a short N-terminal peptideextension that resembles an intact Factor Xa site and a short N-terminalextension due to polylinker sequence (IEGRISEFGS).

Secondly, the DNA encoding a self-splicing intein sequence may beemployed, which intein may be induced to self-splice under pH and/ortemperature control. The intein sequence (represented in SEQ ID 110 asthe polypeptide sequenceISEFRESGAISGDSLISLASTGKRVSIKDLLDEKDFEIWAINEQTMKLESAKVSRVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDELSLKEHIALPRKLESSSLQLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFVANDIIVHN) facilitates self-cleavage ofthe illustrated polypeptide (ie. purification tag-LH_(N)/B) to yield asingle polypeptide molecule with no purification tag. This process doesnot therefore require treatment of the initial expression product withproteases, and the resultant polypeptide (ie. L-chain—Factor Xaactivation site—H_(N)) is simply illustrative of how this approach maybe applied.

According to a further embodiment of the invention, which is describedin an example below, there is provided a polypeptide lacking a portiondesignated H_(C) of a clostridial toxin heavy chain. This portion, seenin the naturally produced toxin, is responsible for binding of toxin tocell surface receptors prior to internalisation of the toxin. Thisspecific embodiment is therefore adapted so that it can not be convertedinto active toxin, for example by the action of a proteolytic enzyme.The invention thus also provides a polypeptide comprising a clostridialtoxin light chain and a fragment of a clostridial toxin heavy chain,said fragment being not capable of binding to those cell surfacereceptors involved in the intoxicating action of clostridial toxin, andit is preferred that such a polypeptide lacks an intact portiondesignated H_(C) of a clostridial toxin heavy chain.

In further embodiments of the invention there are provided compositionscontaining a polypeptide comprising a clostridial toxin light chain anda portion designated H_(N) of a clostridial toxin heavy chain, andwherein the composition is free of clostridial toxin and free of anyclostridial toxin precursor that may be converted into clostridial toxinby the action of a proteolytic enzyme. Examples of these compositionsinclude those containing toxin light chain and H_(N) sequences ofbotulinum toxin types A, B, C₁, D, E, F and G.

The polypeptides of the invention are conveniently adapted to bind to,or include, a third domain (eg. a ligand for targeting to desiredcells). The polypeptide optionally comprises a sequence that binds to,for example, an immunoglobulin. A suitable sequence is a tandem repeatsynthetic IgG binding domain derived from domain B of Staphylococcalprotein A. Choice of immunoglobulin specificity then determines thetarget for a polypeptide—immunoglobulin complex. Alternatively, thepolypeptide comprises a non-clostridial sequence that binds to a cellsurface receptor, suitable sequences including insulin-like growthfactor-1 (IGF-1) which binds to its specific receptor on particular celltypes and the 14 amino acid residue sequence from the carboxy-terminusof cholera toxin A subunit which is able to bind the cholera toxin Bsubunit and thence to GM1 gangliosides. A polypeptide according to theinvention thus, optionally, further comprises a third domain adapted forbinding of the polypeptide to a cell.

According to a second aspect the invention there is provided a fusionprotein comprising a fusion of (a) a polypeptide of the invention asdescribed above with (b) a second polypeptide (also known as apurification tag) adapted for binding to a chromatography matrix so asto enable purification of the fusion protein using said chromatographymatrix. It is convenient for the second polypeptide to be adapted tobind to an affinity matrix, such as a glutathione Sepharose, enablingrapid separation and purification of the fusion protein from an impuresource, such as a cell extract or supernatant.

One possible second purification polypeptide isglutathione-S-transferase (GST), and others will be apparent to a personof skill in the art, being chosen so as to enable purification on achromatography column according to conventional techniques.

According to another embodiment of the present invention, spacersequences may be introduced between two or more domains of the singlechain polypeptide molecule. For example, a spacer sequence may beintroduced between the second and third domains of a polypeptidemolecule of the present invention. Alternatively (or in addition), aspacer sequence may be introduced between a purification tag and thepolypeptide of the present invention or between the first and seconddomains. A spacer sequence may include a proteolytic cleavage site.

In more detail, insertion of a specific peptide sequence into the seconddomain-third domain junction may been performed with the purpose ofspacing the third domain (eg. ligand) from the second domain (eg.H_(N)). This approach may facilitate efficient interaction of the thirddomain with the specific binding target and/or improve the foldingcharacteristics of the polypeptide. Example spacer peptides are providedin Table 2.

TABLE 2 spacer sequences Sequence Illustrated in SEQ ID No (GGGGS)₃39/40, 43/44, 49/50, 53/54, 57/58 RNAse A loop 138/139 Helical 41/42,45/46, 47/48, 51/52, 55/56 Att sites (TSLYKKAGFGS or 133 DPAFLYKV)

In a preferred embodiment, a spacer sequence may be introduced betweenthe first and second domains. For example, a variety of first domain(eg. L-chain) expression constructs have been prepared that incorporatefeatures that are advantageous to the preparation of novel singlepolypeptide hybrid first domain-second domain fusions. Such expressioncassettes are illustrated by SEQ ID NO 69, 71, 73, 75, 77 & 113.

The above cassettes take advantage of a natural linker sequence thatexists in the region between the C-terminus of the L-chain and theN-terminus of the H_(N) domain of a native clostridial neurotoxin. Inmore detail, there is a cysteine at each end of the natural linkersequence that serve to couple the L-chain and H_(N) domain togetherfollowing proteolytic cleavage of the single chain polypeptide moleculeinto its dichain counterpart. These cysteine groups are preserved in theabove-mentioned cassettes. Thus, by maintaining the cysteine amino acidsat either end of the linker sequence, and optionally incorporating aspecific proteolytic site to replace the native sequence, a variety ofconstructs have been prepared that have the property of beingspecifically cleavable between the first and second domains.

For example, by fusing a sequence of interest, such as H_(N)/B to thesequence described in SEQ ID 69, it is possible to routinely prepareL-chain/A-H_(N)/B novel hybrids that are linked through a specificlinker region that facilitates disulphide bond formation. Thus, theexpressed fusion proteins are suitable for proteolytic cleavage betweenthe first (L-chain) and second (H_(N)) domains. The same linkers,optionally including said cleavage site, may be used to link together.other domains of the polypeptide or fusion protein of the presentinvention.

In a further embodiment of the present invention, molecular clamps maybe used to clamp together two or more domains of the polypeptides orfusion proteins of the present invention. Molecular clamps may beconsidered a particular sub-set of the aforementioned spacer sequences.

In more detail, molecular clamping (also known as directed coupling) isa method for joining together two or more polypeptide domains throughthe use of specific complementary peptide sequences that facilitatenon-covalent protein-protein interactions.

Examples of such peptide sequences include leucine zippers (jun & fos),polyionic peptides (eg. poly-glutamate and its poly-arginine pair) andthe synthetic IgG binding domain of Staphylococcal protein A.

Polypeptides comprising first and second domains (eg. LH_(N)) have beenprepared with molecular clamping sequences fused to the C-terminus ofthe second (eg. H_(N)) domain through two methods.

First, DNA encoding the molecular clamp has been ligated directly to theDNA encoding an LH_(N) polypeptide, after removing the STOP codonpresent in the LH_(N) coding sequence. By insertion, to the 3′ of theLH_(N) sequence, of overlapping oligonucleotides encoding the clampsequence and a 3′ STOP codon, an expression cassette has been generated.An example of such a sequence is presented in SEQ ID 63 in which the DNAsequence coding for the molecular clamp known as fos(LTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAH) has been introduced to the 3′of a nucleic acid molecule encoding a LH_(N)/A polypeptide, whichmolecule also has a nucleic acid sequence encoding an enterokinasecleavage site between the coding regions of the first domain (L-chain)and the second domain (H_(N)).

Secondly, site-specific recombination has been utilised to incorporate aclamp sequence to the 3′ of a LH_(N) polypeptide (see, for example, theGATEWAY® cloning system described below) spaced from the H_(N) domain bythe short peptide Gly-Gly. Use of this peptide to space clamp sequencesfrom the C-terminus of H_(N) is illustrated in SEQ 117/118.

In some embodiments, it may be preferable to incorporate cysteine sidechains into the clamp peptide to facilitate formation of disulphidebonds across the clamp, and so make a covalent linkage between the, forexample, second domain (H_(N)) and a third domain (eg. a ligand).Incorporation of the cysteine codon into the clamp sequence has beenperformed by standard techniques, to result in sequences of the typerepresented by SEQ ID 59/60, 61/62, 117/118 and 119/120.

A schematic for the application of molecular clamping to the preparationof suitable LH_(N) polypeptides is illustrated in FIG. 15.

A further alternative for expression of a full-length polypeptidecontaining first and second domains that is suitable for site-specificcoupling to a third domain (eg. a ligand) is to incorporate an inteinself-cleaving sequence into the 3′ of the second domain (eg. H_(N)). SEQID 67/68 illustrates one such construct, in which LH_(N)/A having anenterokinase cleavage site between the first (eg. L-chain) and second(eg. H_(N)) domains is expressed with a Cys residue at the C-terminus,followed by the intein sequence. Following self-cleavage, a reactivethioester is then formed that can take part in a directed couplingreaction to a third domain, for example, as described by Bruick et al,Chem. Biol. (1996), pp. 49-56. Such a polypeptide facilitatessite-specific chemical coupling to third domains (eg. ligands ofinterest) without the problems associated with random derivatisation andrandom coupling which may otherwise result in a heterogenous finalproduct.

As will be appreciated by a skilled person from the entire disclosure ofthe present application, first and second domains may employ L-chain andH-chain components from any clostridial neurotoxin source. Whilstbotulinum sources may be preferred, tetanus sources have equalapplicability. In this respect, the whole sequence of tetanus neurotoxin(TeNT) as published prior to the present application by Eisel, U. et al(1986) EMBO J. 5 (10), pp. 2495-2502, and Accession No. X04436 isincluded in the present application as SEQ ID 140/141 for ease ofreference.

To help illustrate this point, several TeNT based polypeptides have beenprepared according to the present invention, and reference is made toSEQ ID 143 which is an LH_(N) polypeptide having a C-terminal sequenceof EEDIDV₈₇₉. Reference is also made to SEQ ID 147 which is an LH_(N)polypeptide having a C-terminal sequence of EEDIDVILKKSTIL₈₈₇. Both ofthese LH_(N) sequences are representative of ‘native’ TeNT LH_(N)sequences, which have no introduced specific cleavage site between theL-chain and the H_(N) domain. Thus, SEQ ID 145 illustrates a TeNTpolypeptide according to the present invention in which the natural TeNTlinker region between the L-chain and the H_(N) domain has been replacedwith a polypeptide containing a specific enterokinase cleavage sequence.

It will be also appreciated that the general approaches described in thepresent specification for introducing specific cleavage sites andspacer/clamping sequences between any two domains (eg. the L-chain andthe H_(N) domain, or the L-chain and a purification tag) are routinelyapplicable to the preparation of TeNT-containing polypeptide moleculesaccording to the present invention.

A third aspect of the invention provides a composition comprising aderivative of a clostridial toxin, said derivative retaining at least10% of the endopeptidase activity of the clostridial toxin, saidderivative further being non-toxic in vivo due to its inability to bindto cell surface receptors, and wherein the composition is free of anycomponent, such as toxin or a further toxin derivative, that is toxic invivo. The activity of the derivative preferably approaches that ofnatural toxin, and is thus preferably at least 30% and most preferablyat least 60% of natural toxin. The overall endopeptidase activity of thecomposition will, of course, also be determined by the amount of thederivative that is present.

While it is known to treat naturally produced clostridial toxin toremove the H_(C) domain, this treatment does not totally remove toxicityof the preparation, instead some residual toxin activity remains.Natural toxin treated in this way is therefore still not entirely safe.The composition of the invention, derived by treatment of a pure sourceof polypeptide advantageously is free of toxicity, and can convenientlybe used as a positive control in a toxin assay, as a vaccine againstclostridial toxin or for other purposes where it is essential that thereis no residual toxicity in the composition.

The invention enables production of the polypeptides and fusion proteinsof the invention by recombinant means.

A fourth aspect of the invention provides a nucleic acid encoding apolypeptide or a fusion protein according to any of the aspects of theinvention described above.

In one embodiment of this aspect of the invention, a DNA sequenceprovided to code for the polypeptide or fusion protein is not derivedfrom native clostridial sequences, but is an artificially derivedsequence not preexisting in nature.

A specific DNA (SEQ ID NO: 1) described in more detail below encodes apolypeptide or a fusion protein comprising nucleotides encoding residues1-871 of a botulinum toxin type A. Said polypeptide comprises the lightchain domain and the first 423 amino acid residues of the amino terminalportion of a botulinum toxin type A heavy chain. This recombinantproduct is designated LH₄₂₃/A (SEQ ID NO: 2).

In a second embodiment of this aspect of the invention a DNA sequencewhich codes for the polypeptide or fusion protein is derived from nativeclostridial sequences but codes for a polypeptide or fusion protein notfound in nature.

A specific DNA (SEQ ID NO: 19) described in more detail below encodes apolypeptide or a fusion protein and comprises nucleotides encodingresidues 1-1171 of a botulinum toxin type B. Said polypeptide comprisesthe light chain domain and the first 728 amino acid residues of theamino terminal protein of a botulinum type B heavy chain. Thisrecombinant product is designated LH_(728/)B (SEQ ID NO: 20).

The invention thus also provides a method of manufacture of apolypeptide comprising expressing in a host cell a DNA according to thethird aspect of the invention. The host cell is suitably not able tocleave a polypeptide or fusion protein of the invention so as toseparate light and heavy toxin chains; for example, a non-clostridialhost.

The invention further provides a method of manufacture of a polypeptidecomprising expressing in a host cell a DNA encoding a fusion protein asdescribed above, purifying the fusion protein by elution through achromatography column adapted to retain the fusion protein, elutingthrough said chromatography column a ligand adapted to displace thefusion protein and recovering the fusion protein. Production ofsubstantially pure fusion protein is thus made possible. Likewise, thefusion protein is readily cleaved to yield a polypeptide of theinvention, again in substantially pure form, as the second polypeptidemay conveniently be removed using the same type of chromatographycolumn.

The LH_(N)/A derived from dichain native toxin requires extendeddigestion with trypsin to remove the C-terminal ½ of the heavy chain,the H_(C) domain. The loss of this domain effectively renders the toxininactive in vivo by preventing its interaction with host target cells.There is, however, a residual toxic activity which may indicate acontaminating, trypsin insensitive, form of the whole type A neurotoxin.

In contrast, the recombinant preparations of the invention are theproduct of a discreet, defined gene coding sequence and can not becontaminated by full length toxin protein. Furthermore, the product asrecovered from E. coli, and from other recombinant expression hosts, isan inactive single chain peptide or if expression hosts produce aprocessed, active polypeptide it is not a toxin. Endopeptidase activityof LH₄₂₃/A, as assessed by the current in vitro peptide cleavage assay,is wholly dependent on activation of the recombinant molecule betweenresidues 430 and 454 by trypsin. Other proteolytic enzymes that cleavebetween these two residues are generally also suitable for activation ofthe recombinant molecule. Trypsin cleaves the peptide bond C-terminal toArginine or C-terminal to Lysine and is suitable as these residues arefound in the 430-454 region and are exposed (see FIG. 12).

The recombinant polypeptides of the invention are potential therapeuticagents for targeting to cells expressing the relevant substrate butwhich are not implicated in effecting botulism. An example might bewhere secretion of neurotransmitter is inappropriate or undesirable oralternatively where a neuronal cell is hyperactive in terms of regulatedsecretion of substances other than neurotransmitter. In such an examplethe function of the H_(C) domain of the native toxin could be replacedby an alternative targeting sequence providing, for example, a cellreceptor ligand and/or translocation domain.

One application of the recombinant polypeptides of the invention will beas a reagent component for synthesis of therapeutic molecules, such asdisclosed in WO-A-94/21300. The recombinant product will also findapplication as a non-toxic standard for the assessment and developmentof in vitro assays for detection of functional botulinum or tetanusneurotoxins either in foodstuffs or in environmental samples, forexample as disclosed in EP-A-0763131.

A further option is addition, to the C-terminal end of a polypeptide ofthe invention, of a peptide sequence which allows specific chemicalconjugation to targeting ligands of both protein and non-protein origin.

In yet a further embodiment an alternative targeting ligand is added tothe N-terminus of polypeptides of the invention. Recombinant LH_(N)derivatives have been designated that have specific protease cleavagesites engineered at the C-terminus of the LC at the putative trypsinsensitive region and also at the extreme C-terminus of the completeprotein product. These sites will enhance the activational specificityof the recombinant product such that the dichain species can only beactivated by proteolytic cleavage of a more predictable nature than useof trypsin.

The LH_(N) enzymatically produced from native BoNT/A is an efficientimmunogen and thus the recombinant form with its total divorce from anyfull length neurotoxin represents a vaccine component. The recombinantproduct may serve as a basal reagent for creating defined proteinmodifications in support of any of the above areas.

Recombinant constructs are assigned distinguishing names on the basis oftheir amino acid sequence length and their Light Chain (L-chain, L) andHeavy Chain (H-chain, H) content as these relate to translated DNAsequences in the public domain or specifically to SEQ ID NO: 2 and SEQID NO: 20. The ‘LH’ designation is followed by ‘/X’ where ‘X’ denotesthe corresponding clostridial toxin serotype or class, e.g. ‘A’ forbotulinum neurotoxin type A or ‘TeTx’ for tetanus toxin. Sequencevariants from that of the native toxin polypeptide are given inparenthesis in standard fornat, namely the residue position numberprefixed by the residue of the native sequence and suffixed by theresidue of the variant.

Subscript number prefixes indicate an amino-terminal (N-terminal)extension, or where negative a deletion, to the translated sequence.Similarly, subscript number suffixes indicate a carboxy terminal(C-terminal) extension or where negative numbers are used, a deletion.Specific sequence inserts such as protease cleavage sites are indicatedusing abbreviations, e.g. Factor Xa is abbreviated to FXa. L-chainC-terminal suffixes and H-chain N-terminal prefixes are separated by a‘/’ to indicate the predicted junction between the L and H-chains.Abbreviations for engineered ligand sequences are prefixed or suffixedto the clostridial L-chain or H-chain corresponding to their position inthe translation product.

Following this nomenclature,

LH₄₂₃/A = SEQ ID NO: 2, containing the entire L-chain and 423 aminoacids of the H-chain of botulinum neurotoxin type A; ₂LH₄₂₃/A = avariant of this molecule, containing a two amino acid extension to theN-terminus of the L-chain; ₂L_(/2)H₄₂₃/A = a further variant in whichthe molecule contains a two amino acid extension on the N-terminus ofboth the L-chain and the H-chain; ₂L_(FXa/2)H₄₂₃/A = a further variantcontaining a two amino acid extension to the N-terminus of the L-chain,and a Factor Xa cleavage sequence at the C-terminus of the L-chainwhich, after cleavage of the molecule with Factor Xa leaves a two aminoacid N- terminal extension to the H-chain component; and₂L_(FXa/2)H₄₂₃/A-IGF-1 = a variant of this molecule which has a furtherC-terminal extension to the H-chain, in this example the insulin-likegrowth factor 1 (IGF-1) sequence.

The basic molecular biology techniques required to carry out the presentinvention were readily available in the art before the priority date ofthe present application and, as such, would be routine to a skilledperson.

Example 1 of the present application illustrates conventionalrestriction endonuclease-dependent cleavage and ligation methodologiesfor preparing nucleic acid sequences encoding polypeptides of thepresent invention.

Example 4 et seq illustrate a number of alternative conventional methodsfor engineering recombinant DNA molecules that do not requiretraditional methods of restriction endonuclease-dependent cleavage andligation of DNA. One such method is the site-specific recombinationGATEWAY® cloning system of Invitrogen, Inc., which uses phagelambda-based site-specific recombination [Landy, A. (1989) Ann. Rev.Biochem. 58, pp. 913-949]. This method is now described in slightly moredetail.

Using standard restriction endonuclease digestion, or polymerase chainreaction techniques, a DNA sequence encoding first and second domains(e.g. a BoNT LH_(N) molecule) may be cloned into an ENTRY VECTOR(cloning vector). There are a number of options for creation of thecorrect coding region flanked by requisite att site recombinationsequences, as described in the GATEWAY® (cloning system) manual.

For example, one route is to insert a generic polylinker into the ENTRYVECTOR (cloning vector), in which the inserted DNA contains two attsites separated by the polylinker sequence. This approach facilitatesinsertion of a variety of fragments into the ENTRY VECTOR (cloningvector), at user-defined restriction endonuclease sites.

A second route is to insert att sites into the primers used foramplification of the DNA of interest. In this approach, the DNA sequenceof the amplified fragment is modified to include the appropriate aftsites at the 5′ and 3′ ends.

Examples of ENTRY VECTORs (cloning vectors) are provided for LH_(N)/C(SEQ ID 135), for LH_(N)/C with no STOP codon thereby facilitatingdirect fusion to ligands (SEQ ID 136), and for a L-chain/C sequence thatcan facilitate combination with an appropriate second or third domain(SEQ ID 134).

By combination of the modified ENTRY VECTOR (cloning vector) (containingthe DNA of interest) and a DESTINATION VECTOR (cloning vector) ofchoice, an expression clone is generated. The DESTINATION VECTOR(cloning vector) typically provides the necessary information tofacilitate transcription of the inserted DNA of interest and, whenintroduced into an appropriate host cell, facilitates expression ofprotein.

DESTINATION VECTORs (cloning vectors) may be prepared to ensureexpression of N-terminal and/or C-terminal fusion tags and/or additionalprotein domains. An example of a novel engineered DESTINATION VECTOR(cloning vector) for the expression of MBP-tagged proteins in anon-transmissible vector backbone is presented in SEQ ID 137. In thisspecific embodiment, recombination of an ENTRY VECTOR (cloning vector)possessing a sequence of interest with the DESTINATION VECTOR (cloningvector) identified in SEQ ID 137 results in an expression vector for E.coli expression.

The combination of ENTRY VECTORs (cloning vectors) and DESTINATIONVECTORs (cloning vectors) to prepare an expression clone results in anexpressed protein that has a modified sequence. In the Examplesillustrated with SEQ ID 30 & 124, a peptide sequence of TSLYKKAGF is tobe found at the N-terminus of the endopeptidase following cleavage toremove the purification tag. This peptide sequence is encoded by the DNAthat forms the att site and is a feature of all clones that areconstructed and expressed in this way.

It will be appreciated that the att site sequence may be modified toinsert DNA encoding a specific protease cleavage site (for example fromTable 1) to the 3′ of the att site of the entry clone.

It will be also appreciated that the precise N-terminus of anypolypeptide (eg. a LH_(N) fragment) will vary depending on how theendopeptidase DNA was introduced into the ENTRY VECTOR (cloning vector)and its relationship to the 5′ att site. SEQ ID 29/30 & 123/124 are acase in point. The N-terminal extension of SEQ ID 30 is TSLYKKAGFGSwhereas the N-terminal extension of SEQ ID 124 is ITSLYKKAGFGSLDH. Theseamino acid extension-containing domains provide further examples offirst/second domain variants according to the present invention.

The invention also relates to the use of polypeptides according to anyaspect of the invention, or antibodies that bind to these polypeptides,for treating or preventing clostridial neurotoxin poisoning.

The term “treating” includes post-exposure therapy and amelioration oftoxin poisoning. The term “preventing” includes reducing theseverity/intensity of, or the initiation of, toxin poisoning.

Both “treating” and “preventing” encompass the administration to asubject of compositions comprising (i) a polypeptide of the invention,(ii) an antibody that binds to a polypeptide of the invention, (iii) apolynucleotide that encodes a polypeptide of the invention, or acombination thereof.

A particular embodiment of the invention provides for an antigeniccomposition comprising a polypeptide of the invention. In a preferredembodiment, the term “antigenic composition” may be consideredsynonymous with a “vaccine composition”. Also provided is a DNA vaccinecomposition comprising a nucleic acid encoding a polypeptide of theinvention.

The invention also relates to antibodies that bind to polypeptides ofthe invention, and to compositions comprising these antibodies.Preferably, the antibodies bind specifically to the polypeptides.

In a related aspect, there is provided a method of immunising against ortreating clostridial neurotoxin poisoning in a subject comprisingadministering to said subject a therapeutically effective amount of (i)an antigenic composition comprising a polypeptide of the invention, (ii)a composition comprising antibodies of the invention, (iii) a DNAvaccine composition comprising a nucleic acid encoding a polypeptide ofthe invention, or a combination thereof.

In compositions of the invention, the immunogenicity of the epitopes ofthe polypeptides may be enhanced by preparing them in mammalian or yeastsystems fused with or assembled with particle-forming proteins such as,for example, that associated with hepatitis B surface antigen. Thecompositions may be prepared from one or more peptides of the invention.

Typically, compositions of the invention are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. The preparation may also be emulsified, or the peptideencapsulated in liposomes. The active immunogenic ingredients are oftenmixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, and/or adjuvants which enhancethe effectiveness of the vaccine. Examples of adjuvants which may beeffective include but are not limited to: aluminium hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE),and RIBI, which contains three components extracted from bacteria,monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton(MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.

The compositions are conventionally administered parenterally, byinjection, for example, either subcutaneously or intramuscularly.Additional formulations which are suitable for other modes ofadministration include suppositories. For suppositories, traditionalbinders and carriers may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Thecompositions may take the form of solutions, suspensions, sustainedrelease formulations or powders and typically contain by weight 10%-95%of active ingredient, preferably 25%-70%.

The polypeptides may be formulated into a composition as neutral or saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or with organic acids such as acetic, oxalic, tartaric, maleic, and thelike. Salts formed with the free carboxyl groups may also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

There are a wide variety of approaches to successfully formulating a DNAvaccine. Overall approaches to the preparation and formulation of DNAvaccines are to be found in standard texts such as DNA Vaccines: Methodsand Protocols, edited by D. B. Lowrie & R. G. Whalen, Humana Press,2000, and in specialised journal articles such as Sasaki et al., 2003,Adjuvant formulations and delivery systems for DNA vaccines, Methods 31,243-254. For a review of the mechanism of action of DNA vaccines seeLeitner et al, 2000, Vaccine, 18, 765-777. Further details of DNAvaccine formulations can be found in Example 21. The mechanism of actionof a DNA vaccine is well known. In brief, however, a DNA encoding apolypeptide of the invention is injected into a host, typically into themuscle cells, where it leads to the synthesis of the polypeptide insidethe host Tells. Before injection, the DNA is usually modified tocomprise a gene promoter that is appropriate to the particular host. Thegeneration of foreign polypeptides within the host cells results in astrong immune response; killer T cells, as well as circulatingantibodies, are generated.

The compositions of the invention are administered in a mannercompatible with the dosage formulation, and in such amount as will beprophylactically and/or therapeutically effective. The quantity to beadministered, which is generally in the range of 5 micrograms to 250micrograms of antigen per dose, depends on the subject to be treated,capacity of the subject's immune system to synthesize antibodies, andthe degree of protection desired. Precise amounts of active ingredientrequired to be administered may depend on the judgment of thepractitioner and may be peculiar to each subject.

The compositions may be given in a single dose schedule, or preferablyin a multiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand or re-enforce the immune response, for example, at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by theneed of the individual and be dependent upon the judgment of thepractitioner.

In addition, compositions containing polypeptide may be administered inconjunction with other immunoregulatory agents, for example,immunoglobulins, as well as antibiotics.

The compositions may be administered by conventional routes, eg.intravenous, intraperitoneal, intranasal routes.

As described above, polypeptides of the invention or derivatives,variants, fragments, analogs or homologs thereof, may be utilized asimmunogens in the generation of antibodies that bind, preferablyspecifically, to these protein components. The term “antibody” as usedherein refers to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantigen binding site that specifically binds (immunoreacts with) anantigen. Such antibodies include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, F_(ab) and F_((ab′)2) fragments, andan F_(ab) expression library. Various procedures known within the artmay be used for the production of polyclonal or monoclonal antibodies topolypeptides of the invention, or derivatives, fragments, analogs orhomologs thereof. Some of these proteins are discussed below.

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byinjection with the native protein, or a synthetic variant thereof, or aderivative of the foregoing. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed polypeptides or chemicallysynthesized polypeptides. The preparation can further include anadjuvant. Various adjuvants used to increase the immunological responseinclude, but are not limited to, Freund's (complete and incomplete),mineral gels (e.g., aluminium hydroxide), surface active substances(e.g., lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, dinitrophenol, etc), human adjuvants such as BacilleCalmatte-Guerin and Corynebacterium parvum, or similar immunostimulatoryagents. If desired, the antibody molecules directed against thepolypeptides can be isolated from the mammal (e.g. from the blood) andfurther purified by well known techniques, such as protein Achromatography to obtain the IgG fraction.

The term “monoclonal antibody” or “monoclonal antibody composition”, asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of the polypeptides of the invention. Amonoclonal antibody composition thus typically displays a single bindingaffinity for a particular polypeptide of the invention with which itimmunoreacts. For preparation of monoclonal antibodies directed towardsa particular polypeptide or derivatives, fragments, analogs or homologsthereof, any technique that provides for the production of antibodymolecules by continuous cell line culture may be utilized. Suchtechniques include, but are not limited to, hybridoma technique (seeKohler & Milstein, 1975 Nature 256: 495-497); the trioma technique; thehuman B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today4:72) and the EBV hybridoma technique to produce human monoclonalantibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies maybe utilized in the practice of the present invention and may be producedby using human hybridomas (see Cote, et al., 1983. Proc Natl Acad ScieUSA 80: 2026-2030) or by transforming human B-cells with Epstein BarrVirus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES ANDCANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to a polypeptide of the invention(see e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adaptedfor the construction of F_(ab) expression libraries (see e.g., Huse, etal. 1989 Science 246: 1275-1281) to allow rapid and effectiveidentification of monoclonal F_(ab) fragments with the desiredspecificity for a polypeptide of the invention or a derivative,fragment, analog or homolog thereof. Non-human antibodies can be“humanized” by techniques well known in the art. See e.g., U.S. Pat. No.5,225,539. Antibody fragments that contain the idiotypes may be producedby techniques known in the art including, but not limited to: (i) anF_((ab′)2) fragment produced by pepsin digestion of an antibodymolecule; (ii) an F_(ab) fragment generated by reducing the disulfidebridges of an F_((ab′)2) fragment; (iii) fragment generated by thetreatment of the antibody molecule with papain and a reducing agent and(iv) F_(v) fragments.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT InternationalApplication No. PCT/US86/02269; European Patent Application No. 184,187;European Patent Application No. 171,496; European Patent Application No.173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No.4,816,567; European Patent Application No. 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443: Liu et al.(1987) J Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218;Nishimura et al. (1987) Cancer Res 47:999-1005; Wood et al. (1985)Nature 214:446-449; Shaw et al. (1988) J Natl Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler etal. (1988) J Immunol 141-4053-4060.

In one embodiment, methods for the screening of antibodies that possessthe desired specificity include, but are not limited to, enzyme-linkedimmunosorbent assay (ELISA) and other immunologically-mediatedtechniques known within the art. In a specific embodiment, selection ofantibodies that are specific to a particular domain of a polypeptide ofthe invention are facilitated by generation of hybridomas that bind tothe fragment of a polypeptide of the invention possessing such a domain.Antibodies that are specific for one or more domains within apolypeptide of the invention are also provided herein.

The antibodies of the invention, or compositions comprising theantibodies, may be usefully employed in “passive therapy”, where animmediate state of immunisation is created by the post-exposureintroduction of antibodies into a patient. Typically, compositions ofthe invention are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The composition maycontain excipients which are pharmaceutically acceptable and compatiblewith the antibodies. Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. In addition, if desired, the composition may contain minoramounts of auxiliary substances such as wetting or emulsifying agentsand pH buffering agents. The compositions are conventionallyadministered parenterally, by injection, for example, eithersubcutaneously or intramuscularly, in a manner compatible with thedosage formulation, and in such amount as will be prophylacticallyand/or therapeutically effective. Precise amounts of the compositionrequired to be administered may depend on the judgment of thepractitioner and may be peculiar to each subject. Further details ofpassive therapy formulations are described in Example 22.

Examples of the use of passive therapy in the botulinum field can befound in e.g. Arturo Casadevall, Passive Antibody Administration(Immediate Immunity) as a Specific Defense against Biological WeaponsEmerg Infect Dis [serial online] 2002 Aug.; 8; Mowry M C et al.Production and purification of a chimeric monoclonal antibody againstbotulinum neurotoxin serotype A. Protein Expr Purif. 2004 October;37(2):399-408; Nowakowski A et al. Potent neutralization of botulinumneurotoxin by recombinant oligoclonal antibody. Proc Natl Acad Sci USA.2002 Aug. 20; 99(17):11346-50.

The experiments described in the Examples section of this applicationdemonstrate the utility of polypeptides, antibodies and polynucleotidesof the invention in the prevention and treatment of clostridial toxinpoisoning.

In experiments carried out by the present inventors, antisera againstLH_(N)/A at a dilution of 1:40 protected mice in i.p. challenge with upto 10⁴ MLD₅₀ (=0.05 micrograms) of BoNT/A. Further support for theutility of the polypeptides and antibodies is provided by experimentsdescribed in Chaddock et al 2002 (Protein Expression & Purification, 25,219-228), the contents of which is incorporated herein by reference. Inthese experiments, incubation of anti-recLH_(N)/A antisera with 3.2×10⁴LD₅₀ BoNT/A inhibited BoNT/A-mediated death of all the animals withinthe test group. In contrast, incubation of a similar quantity ofpre-immune serum with only 1.6×10¹ LD₅₀ BoNT/A resulted in no survivinganimals. Antisera raised to the LC and H_(N) domains thereforeeffectively inhibited BoNT/A holotoxin-mediated toxic effects.

The BoNT/A neutralising ability of anti-LH_(N)/A antisera is alsodemonstrated by in vitro experiments described in Hall et al 2004(Journal Immunological Methods, 288, 55-60), the contents of which isincorporated herein by reference. In a first set of experiments, theability of anti-BoNT antisera, anti-H_(C) antisera, and anti-recLH_(N)/Aantisera to neutralise the effects of BoNT/A was assessed using the eSCNmodel. Anti-recLH_(N)/A antisera was shown to fully inhibit the effectsof 3 pM BoNT/A in the in vitro eSCN assay when mixed at a 10000-molarexcess of antibody.

In a second set of experiments, the ability of anti-recLH_(N)/A antiserato neutralise the effects of BoNT/A, B and C was assessed using the eSCNmodel. In these experiments, no neutralising effect was observed withBoNT/B or BoNT/C (at similar antibody:toxin excesses as used in thefirst set of experiments) suggesting that the protective effect of theantibody may be serotype specific.

Other experiments confirming the effectiveness of LH_(N) fragments asvaccine components include the challenge studies performed by Jensen etal 2003 (Toxicon, 41, 691-701), the contents of which is incorporatedherein by reference. In these studies, mice were inoculated with adefined quantity of antigen (e.g. LC/A, LH_(N)/A) before subsequentchallenge with defined concentrations of BoNT/A. Mice inoculated with 5μg or 15 μg LC/A were 100% susceptible to challenge with 10³ or 10⁴MLD₅₀ BoNT/A. However, mice inoculated with 5 μg or 15 μg LH_(N)/A were100% protected to challenge with 10³ or 10⁴ MLD₅₀ BoNT/A. Thisdemonstrates the superior antigenic potential of LH_(N)/A compared toLC/A.

There now follows description of specific embodiments of the invention,illustrated by drawings in which:

FIG. 1 shows a schematic representation of the domain structure ofbotulinum neurotoxin type A (BoNT/A);

FIG. 2 shows a schematic representation of assembly of the gene for anembodiment of the invention designated Lh₄₂₃/A;

FIG. 3 is a graph comparing activity of native toxin, trypsin generated“native” LH_(N)/A and an embodiment of the invention designated ₂LH₄₂₃/A(Q₂E,N₂₆K,A₂₇Y) in an in vitro peptide cleavage assay;

FIG. 4 is a comparison of the first 33 amino acids in publishedsequences of native toxin and embodiments of the invention;

FIG. 5 shows the transition region of an embodiment of the inventiondesignated L/₄H₄₂₃/A illustrating insertion of four amino acids at theN-terminus of the H_(N) sequence; amino acids coded for by the Eco 47III restriction endonuclease cleavage site are marked and the H_(N)sequence then begins ALN . . . ;

FIG. 6 shows the transition region of an embodiment of the inventiondesignated L_(Fxa/3)H₄₂₃/A illustrating insertion of a Factor Xacleavage site at the C-terminus of the L-chain, and three additionalamino acids coded for at the N-terminus of the H-sequence; theN-terminal amino acid of the cleavage-activated H_(N) will be cysteine;

FIG. 7 shows the C-terminal portion of the amino acid sequence of anembodiment of the invention designated L_(FXa/3)H₄₂₃/A-IGF-1, a fusionprotein; the IGF-1 sequence begins at position G₈₈₂;

FIG. 8 shows the C-terminal portion of the amino acid sequence of anembodiment of the invention designated L_(FXa/3)H₄₂₃/A-CtxA14, a fusionprotein; the C-terminal CtxA sequence begins at position Q₈₈₂;

FIG. 9 shows the C-terminal portion of the amino acid sequence Omanembodiment of the invention designated L_(FXa/3)H₄₂₃/A-ZZ, a fusionprotein; the C-terminal ZZ sequence begins at position A₈₉₀ immediatelyafter a genenase recognition site (underlined);

FIGS. 10 & 11 show schematic representations of manipulations ofpolypeptides of the invention; FIG. 10 shows Lh₄₂₃/A with N-terminaladdition of an affinity purification peptide (in this case GST) andC-terminal addition of an Ig binding domain; protease cleavage sites R1,R2 and R3 enable selective enzymatic separation of domains; FIG. 11shows specific examples of protease cleavage sites R1, R2 and R3 and aC-terminal fusion peptide sequence;

FIG. 12 shows the trypsin sensitive activation region of a polypeptideof the invention;

FIG. 13 shows Westem blot analysis of recombinant LH₁₀₇/B expressed fromE coli; panel A was probed with anti-BoNT/B antiserum; Lane 1, molecularweight standards; lanes 2 & 3, native BoNT/B; lane 4, immunopurifiedLH₁₀₇/B; panel B was probed with anti-T7 peptide tag antiserum; lane 1,molecular weight standards; lanes 2 & 3, positive control E.coli T7expression; lane 4 immunopurified LH₁₀₇/B.

FIG. 14 illustrates a fusion protein of the present invention, whichfusion protein includes two different proteolytic cleavage sites (E1,and E2) between a purification tag (TAG) and a first domain (L-chain),and a duplicate proteolytic cleavage sites (E2) between a first domain(L-chain) and a second domain (H_(N)). Use of the E2 protease results insimultaneous cleavage at the two defined E2 cleavage sites leaving adichain polypeptide molecule comprising the first and second domains,whereas use of the E1 protease results in cleavage at the single definedE1 cleavage site leaving a single polypeptide chain molecule comprisingthe first and second domains.

FIG. 15 illustrates the use of molecular-clamping technology to fusetogether a polypeptide comprising first and second domains (eg. LH_(N)),and a second molecule comprising a third domain (eg. a ligand);

FIG. 16 shows the BoNT/A neutralising ability of anti-LH_(N)/A;

FIG. 17 shows the ability of anti-recLH_(N)/A to neutralise the effectsof BoNT/A, B and C;

FIG. 18 a shows the ability of anti-recLH_(N)/A antisera to neutralisethe effects of WGA-LH_(N)/A;

FIG. 18 b shows the ability of anti-H_(c) antisera to neutralise theeffects of WGA-LH_(N)/A.

The sequence listing that accompanies this application contains thefollowing sequences:

SEQ ID NO: Sequence 1 DNA coding for LH₄₂₃/A 2 LH₄₂₃/A 3 DNA coding for₂₃LH₄₂₃/A (Q₂E, N₂₆K, A₂₇Y), of which an N-terminal portion is shown inFIG. 4. 4 ₂₃LH₄₂₃/A (Q₂E, N₂₆K, A₂₇Y) 5 DNA coding for ₂LH₄₂₃/A (Q₂E,N₂₆K, A₂₇Y), of which an N-terminal portion is shown in FIG. 4. 6₂LH₄₂₃/A (Q₂E, N₂₆K, A₂₇Y) 7 DNA coding for native BoNT/A according toBinz et al 8 native BoNT/A according to Binz et al 9 DNA coding forL_(/4)H₄₂₃/A 10 L_(/4)H₄₂₃/A 11 DNA coding for L_(FXa)/₃H₄₂₃/A 12L_(FXa)/₃H₄₂₃/A 13 DNA coding for L_(FXa)/₃H₄₂₃/A-IGF-1 14L_(Fxa)/₃H₄₂₃/A-IGF-1 15 DNA coding for L_(FXa)/₃H423/A-CtxA14 16L_(FXa)/₃H₄₂₃/A-CtxA14 17 DNA coding for L_(FXa/3)H₄₂₃/A-ZZ 18L_(FXa/3)H₄₂₃/A-ZZ 19 DNA coding for LH₇₂₈/B 20 LH₇₂₈/B 21 DNA codingfor LH₄₁₇/B 22 LH₄₁₇/B 23 DNA coding for LH₁₀₇/B 24 LH₁₀₇/B 25 DNAcoding for LH₄₂₃/A (Q₂E, N₂₆K, A₂₇Y) 26 LH₄₂₃/A (Q₂E, N₂₆K, A₂₇Y) 27 DNAcoding for LH₄₁₇/B wherein the first 274 bases are modified to have anE. coli codon bias 28 DNA coding for LH₄₁₇/B wherein bases 691-1641 ofthe native BoNT/B sequence have been replaced by a degenerate DNA codingfor amino acid residues 231-547 of the native BoNT/B polypeptide 29 DNAcoding for LH_(N)/A as expressed from a GATEWAY ® (cloning system)adapted DESTINATION VECTOR (cloning vector). LH_(N)/A incorporates anenterokinase activation site at the LC-H_(N) junction and an 11 aminoacid att site peptide extension at the 5′ end of the LH_(N)/A sequence30 LH_(N)/A produced by expression of SEQ ID 29, said polypeptideincorporating an enterokinase activation site at the LC-H_(N) junctionand an 11 amino acid att site peptide extension at the N-terminus of theLH_(N)/A sequence 31 DNA coding for LH_(N)/A with an enterokinaseactivation site at the LC-H_(N) junction 32 LH_(N)/A produced byexpression of SEQ ID 31, said polypeptide having an enterokinaseactivation site at the LC-H_(N) junction 33 DNA coding for LH_(N)/A witha Factor Xa protease activation site at the LC-H_(N) junction 34LH_(N)/A produced by expression of SEQ ID 33, said polypeptide having aFactor Xa protease activation site at the LC-H_(N) junction 35 DNAcoding for LH_(N)/A with a Precission protease activation site at theLC-H_(N) junction 36 LH_(N)/A produced by expression of SEQ ID 35, saidpolypeptide having a Precission protease activation site at the LC-H_(N)junction 37 DNA coding for LH_(N)/A with a Thrombin protease activationsite at the LC-H_(N) junction 38 LH_(N)/A produced by expression of SEQID 37, said polypeptide having a Thrombin protease activation site atthe LC-H_(N) junction 39 DNA coding for an LH_(N)/A-ligand (Erythrinacristagalli lectin) fusion in which the LC-H_(N) junction does notincorporate a specific protease cleavage site and the ligand is spacedfrom the H_(N) domain by a (GGGGS)₃ spacer. 40 LH_(N)/A-ligand(Erythrina cristagalli lectin) fusion produced by expression of SEQ ID39, in which the LC-H_(N) junction does not incorporate a specificprotease cleavage site and the ligand is spaced from the H_(N) domain bya (GGGGS)₃ spacer. 41 DNA coding for LH_(N)/A-ligand (Erythrinacristagalli lectin) fusion in which the LC-H_(N) junction does notincorporate a specific protease cleavage site and the ligand is spacedfrom the H_(N) domain by a helical spacer. 42 LH_(N)/A-ligand (Erythrinacristagalli lectin) fusion produced by expression of SEQ ID 41, in whichthe LC-H_(N) junction does not incorporate a specific protease cleavagesite and the ligand is spaced from the H_(N) domain by a helical spacer.43 DNA coding for LH_(N)/A-ligand (Erythrina cristagalli lectin) fusionin which the LC-H_(N) junction incorporates a specific enterokinaseprotease activation site and the ligand is spaced from the H_(N) domainby a (GGGGS)₃ spacer. 44 LH_(N)/A-ligand (Erythrina cristagalli lectin)fusion produced by expression of SEQ ID 43, in which the LC-H_(N)junction incorporates a specific enterokinase protease activation siteand the ligand is spaced from the H_(N) domain by a (GGGGS)₃ spacer. 45DNA coding for LH_(N)/A-ligand (Erythrina cristagalli lectin) fusion inwhich the LC-H_(N) junction incorporates a specific enterokinaseprotease activation site and the ligand is spaced from the H_(N) domainby a helical spacer. 46 LH_(N)/A-ligand (Erythrina cristagalli lectin)fusion produced by expression of SEQ ID 45, in which the LC-H_(N)junction incorporates a specific enterokinase protease activation siteand the ligand is spaced from the H_(N) domain by a helical spacer. 47DNA coding for LH_(N)/A-ligand (Erythrina cristagalli lectin) fusion inwhich the LC-H_(N) junction incorporates a specific Thrombin proteaseactivation site and the ligand is spaced from the H_(N) domain by ahelical spacer. 48 LH_(N)/A-ligand (Etythrina cristagalli lectin) fusionproduced by expression of SEQ ID 47, in which the LC-H_(N) junctionincorporates a specific Thrombin protease activation site and the ligandis spaced from the H_(N) domain by a helical spacer. 49 DNA coding forLH_(N)/A-ligand (Erythrina cristagalli lectin) fusion in which theLC-H_(N) junction incorporates a specific Thrombin protease activationsite and the ligand is spaced from the H_(N) domain by a (GGGGS)₃spacer. 50 LH_(N)/A-ligand (Erythrina cristagalli lectin) fusionproduced by expression of SEQ ID 49, in which the LC-H_(N) junctionincorporates a specific Thrombin protease activation site and the ligandis spaced from the H_(N) domain by a (GGGGS)₃ spacer. 51 DNA coding forLH_(N)/A-ligand (Erythrina cristagalli lectin) fusion in which theLC-H_(N) junction incorporates a specific Precission protease activationsite and the ligand is spaced from the H_(N) domain by a helical spacer.52 LH_(N)/A-ligand (Erythrina cristagalli lectin) fusion produced byexpression of SEQ ID 51, in which the LC-H_(N) junction incorporates aspecific Precission protease activation site and the ligand is spacedfrom the H_(N) domain by a helical spacer. 53 DNA coding forLH_(N)/A-ligand (Erythrina cristagalli lectin) fusion in which theLC-H_(N) junction incorporates a specific Precission protease activationsite and the ligand is spaced from the H_(N) domain by a (GGGGS)₃spacer. 54 LH_(N)/A-ligand (Erythrina cristagalli lectin) fusionproduced by expression of SEQ ID 53, in which the LC-H_(N) junctionincorporates a specific Precission protease activation site and theligand is spaced from the H_(N) domain by a (GGGGS)₃ spacer. 55 DNAcoding for LH_(N)/A-ligand (Erythrina cristagalli lectin) fusion inwhich the LC-H_(N) junction incorporates a specific Factor Xa proteaseactivation site and the ligand is spaced from the H_(N) domain by ahelical spacer. 56 LH_(N)/A-ligand (Erythrina cristagalli lectin) fusionproduced by expression of SEQ ID 55, in which the LC-H_(N) junctionincorporates a specific Factor Xa protease activation site and theligand is spaced from the H_(N) domain by a helical spacer. 57 DNAcoding for LH_(N)/A-ligand (Erythrina cristagalli lectin) fusion inwhich the LC-H_(N) junction incorporates a specific Factor Xa proteaseactivation site and the ligand is spaced from the H_(N) domain by a(GGGGS)₃ spacer. 58 LH_(N)/A-ligand (Erythrina cristagalli lectin)fusion produced by expression of SEQ ID 57, in which the LC-H_(N)junction incorporates a specific Factor Xa protease activation site andthe ligand is spaced from the H_(N) domain by a (GGGGS)₃ spacer. 59 DNAcoding for LH_(N)/A incorporating an enterokinase protease activationsite at the LC-H_(N) junction and a C- terminal fos ligand bounded by apair of Cys residues 60 LH_(N)/A produced by expression of SEQ ID 59,said polypeptide incorporating an enterokinase protease activation siteat the LC-H_(N) junction and a C-terminal fos ligand bounded by a pairof Cys residues 61 DNA coding for LH_(N)/A incorporating an enterokinaseprotease activation site at the LC-H_(N) junction and a C- terminal(Glu)₈ peptide bounded by a pair of Cys residues 62 LH_(N)/A produced byexpression of SEQ ID 61, said polypeptide incorporating an enterokinaseprotease activation site at the LC-H_(N) junction and a C-terminal(Glu)₈ peptide bounded by a pair of Cys residues 63 DNA coding forLH_(N)/A incorporating an enterokinase protease activation site at theLC-H_(N) junction and a C- terminal fos ligand 64 LH_(N) /A produced byexpression of SEQ ID 63, said polypeptide incorporating an enterokinaseprotease activation site at the LC-H_(N) junction and a C-terminal fosligand 65 DNA coding for LH_(N)/A incorporating an enterokinase proteaseactivation site at the LC-H_(N) junction and a C- terminal (Glu)₈peptide 66 LH_(N)/A produced by expression of SEQ ID 65, saidpolypeptide incorporating an enterokinase protease activation site atthe LC-H_(N) junction and a C-terminal (Glu)₈ peptide 67 DNA coding forLH_(N)/A incorporating an enterokinase protease activation site at theLC-H_(N) junction and a C- terminal self-cleavable intein polypeptide tofacilitate thioester formation for use in chemical directed coupling 68LH_(N)/A produced by expression of SEQ ID 67, said polypeptideincorporating an enterokinase protease activation site at the LC-H_(N)junction and a C-terminal self- cleavable intein polypeptide tofacilitate thioester formation for use in chemical directed coupling 69DNA coding for LC/A with no STOP codon, a linker peptide incorporatingthe first 6 amino acids of the H_(N) domain and an enterokinase cleavagesite. 70 LC/A produced by expression of SEQ ID 69, said polypeptidehaving no STOP codon, a linker peptide incorporating the first 6 aminoacids of the H_(N) domain and an enterokinase cleavage site. 71 DNAcoding for LC/A with no STOP codon, a linker peptide incorporating thefirst 6 amino acids of the H_(N) domain and an Factor Xa cleavage site.72 LC/A produced by expression of SEQ ID 71, said polypeptide having noSTOP codon, a linker peptide incorporating the first 6 amino acids ofthe H_(N) domain and an Factor Xa cleavage site. 73 DNA coding for LC/Awith no STOP codon and a linker peptide representing the native LC-H_(N)sequence incorporating the first 6 amino acids of the H_(N) domain 74LC/A produced by expression of SEQ ID 73, said polypeptide having noSTOP codon and a linker peptide representing the native LC-H_(N)sequence incorporating the first 6 amino acids of the H_(N) domain 75DNA coding for LC/A with no STOP codon, a linker peptide incorporatingthe first 6 amino acids of the H_(N) domain and an Precission cleavagesite. 76 LC/A produced by expression of SEQ ID 75, said polypeptidehaving no STOP codon, a linker peptide incorporating the first 6 aminoacids of the H_(N) domain and an Precission cleavage site. 77 DNA codingfor LC/A with no STOP codon, a linker peptide incorporating the first 6amino acids of the H_(N) domain and an Thrombin cleavage site. 78 LC/Aproduced by expression of SEQ ID 77, said polypeptide having no STOPcodon, a linker peptide incorporating the first 6 amino acids of theH_(N) domain and an Thrombin cleavage site. 79 DNA coding for LH_(N)/Bincorporating an enterokinase protease activation site at the LC-H_(N)junction (in which there are 11 amino acids between the Cys residues ofthe LC & H_(N) domains) and a 6 amino acid N-terminal extension 80LH_(N)/B produced by expression of SEQ ID 79, said polypeptideincorporating an enterokinase protease activation site at the LC-H_(N)junction (in which there are 11 amino acids between the Cys residues ofthe LC & H_(N) domains) and a 6 amino acid N-terminal extension 81 DNAcoding for LH_(N)/B incorporating an enterokinase protease activationsite at the LC-H_(N) junction (in which there are 20 amino acids betweenthe Cys residues of the LC & H_(N) domains) and a 6 amino acidN-terminal extension 82 LH_(N)/B produced by expression of SEQ ID 82,said polypeptide incorporating an enterokinase protease activation siteat the LC-H_(N) junction (in which there are 20 amino acids between theCys residues of the LC & H_(N) domains) and a 6 amino acid N-terminalextension 83 DNA coding for LH_(N)/B incorporating a Factor Xa proteaseactivation site at the LC-H_(N) junction and an 11 amino acid N-terminalextension resulting from cleavage at an intein self-cleaving polypeptide84 LH_(N)/B produced by expression of SEQ ID 83, said polypeptideincorporating a Factor Xa protease activation site at the LC-H_(N)junction and an 11 amino acid N-terminal extension resulting fromcleavage at an intein self-cleaving polypeptide 85 DNA coding forLH_(N)/B incorporating a Factor Xa protease activation site at theLC-H_(N) junction and an 11 amino acid N-terminal extension (retaining aFactor Xa protease cleavage site) resulting from cleavage at a TEVprotease cleavage site (included to release the LH_(N)/B from apurification tag). 86 LH_(N)/B produced by expression of SEQ ID 85, saidpolypeptide incorporating a Factor Xa protease activation site at theLC-H_(N) junction and an 11 amino acid N-terminal extension (retaining aFactor Xa protease cleavage site) resulting from cleavage at a TEVprotease cleavage site (included to release the LH_(N)/B from apurification tag). 87 DNA coding for LH_(N)/B incorporating a Factor Xaprotease activation site at the LC-H_(N) junction and a 6 amino acid N-terminal extension 88 LH_(N)/B produced by expression of SEQ ID 87, saidpolypeptide incorporating a Factor Xa protease activation site at theLC-H_(N) junction and a 6 amino acid N-terminal extension 89 DNA codingfor LH_(N)/B incorporating a Factor Xa protease activation site at theLC-H_(N) junction and an 11 amino acid N-terminal extension (retainingan enterokinase protease cleavage site) resulting from cleavage at aFactor Xa protease cleavage site (included to release the LH_(N)/B froma purification tag). 90 LH_(N)/B produced by expression of SEQ ID 89,said polypeptide incorporating a Factor Xa protease activation site atthe LC-H_(N) junction and an 11 amino acid N-terminal extension(retaining an enterokinase protease cleavage site) resulting fromcleavage at a Factor Xa protease cleavage site (included to release theLH_(N)/B from a purification tag). 91 DNA coding for LH_(N)/Bincorporating a Factor Xa protease activation site at the LC-H_(N)junction and an 10 amino acid N-terminal extension (retaining a FactorXa protease cleavage site) resulting from cleavage at an enterokinaseprotease cleavage site (included to release the LH_(N)/B from apurification tag). 92 LH_(N)/B produced by expression of SEQ ID 91, saidpolypeptide incorporating a Factor Xa protease activation site at theLC-H_(N) junction and an 10 amino acid N-terminal extension (retaining aFactor Xa protease cleavage site) resulting from cleavage at anenterokinase protease cleavage site (included to release the LH_(N)/Bfrom a purification tag). 93 DNA coding for LH_(N)/B incorporating aFactor Xa protease activation site at the LC-H_(N) junction and a 2amino acid (Gly-Ser) N-terminal extension as expressed in pGEX-4T-2 94LH_(N)/B produced by expression of SEQ ID 93, said polypeptideincorporating a Factor Xa protease activation site at the LC-H_(N)junction and a 2 amino acid (Gly-Ser) N- terminal extension as expressedin pGEX-4T-2 95 DNA coding for LH_(N)/B incorporating a Factor Xaprotease activation site at the LC-H_(N) junction and a 7 amino acid(Ser-Pro-Gly-Ala-Arg-Gly-Ser) N-terminal extension as expressed inpET-43a 96 LH_(N)/B produced by expression of SEQ ID 95, saidpolypeptide incorporating a Factor Xa protease activation site at theLC-H_(N) junction and a 7 amino acid (Ser-Pro-Gly- Ala-Arg-Gly-Ser)N-terminal extension as expressed in pET- 43a 97 DNA coding for LH_(N)/Bincorporating a Factor Xa protease activation site at the LC-H_(N)junction and a 7 amino acid (Ala-Met-Ala-Glu-Ile-Gly-Ser) N-terminalextension as expressed in pET-32a 98 LH_(N)/B produced by expression ofSEQ ID 97, said polypeptide incorporating a Factor Xa proteaseactivation site at the LC-H_(N) junction and a 7 amino acid(Ala-Met-Ala- Asp-Ile-Gly-Ser) N-terminal extension as expressed in pET-32a 99 DNA coding for LH_(N)/B incorporating a Thrombin proteaseactivation site at the LC-H_(N) junction and a 6 amino acid (Ile-Ser-Glu-Phe-Gly-Ser) N-terminal extension as expressed in pMAL-c2 100LH_(N)/B produced by expression of SEQ ID 99, said polypeptideincorporating a Thrombin protease activation site at the LC-H_(N)junction and a 6 amino acid (Ile-Ser-Glu- Phe-Gly-Ser) N-terminalextension as expressed in pMAL- c2 101 DNA coding for LH_(N)/Bincorporating a TEV protease activation site at the LC-H_(N) junctionand a 6 amino acid (Ile- Ser-Glu-Phe-Gly-Ser) N-terminal extension asexpressed in pMAL-c2 102 LH_(N)/B produced by expression of SEQ ID 101,said polypeptide incorporating a TEV protease activation site at theLC-H_(N) junction and a 6 amino acid (Ile-Ser-Glu-Phe- Gly-Ser)N-terminal extension as expressed in pMAL-c2 103 DNA coding for LH_(N)/Bincorporating a Factor Xa protease activation site at the LC-H_(N)junction and a 6 amino acid (Ile- Ser-Glu-Phe-Gly-Ser) N-terminalextension as expressed in pMAL-c2. DNA incorporates Mfel and Avrllrestriction enzyme sites for incorporation of novel linker sequences atthe LC-H_(N) junction. 104 LH_(N)/B produced by expression of SEQ ID103, said polypeptide incorporating a Factor Xa protease activation siteat the LC-H_(N) junction and a 6 amino acid (Ile-Ser-Glu- Phe-Gly-Ser)N-terminal extension as expressed in pMAL c2. 105 DNA coding forLH_(N)/B incorporating an enterokinase protease activation site at theLC-H_(N) junction (in which there are 20 amino acids between the Cysresidues of the LC & H_(N) domains) and a 6 amino acid (Ile-Ser-Glu-Phe-Gly-Ser) N-terminal extension. Avrll restriction site is deleted. 106LH_(N)/B produced by expression of SEQ ID 105, said polypeptideincorporating an enterokinase protease activation site at the LC-H_(N)junction (in which there are 20 amino acids between the Cys residues ofthe LC & H_(N) domains) and a 6 amino acid (Ile-Ser-Glu-Phe-Gly-Ser) N-terminal extension 107 DNA coding for LH_(N)/B incorporating anenterokinase protease activation site at the LC-H_(N) junction (in whichthere are 20 amino acids between the Cys residues of the LC & H_(N)domains) and a 6 amino acid (Ile-Ser-Glu-Phe- Gly-Ser) N-terminalextension. 108 LH_(N)/B produced by expression of SEQ ID 107, saidpolypeptide incorporating an enterokinase protease activation site atthe LC-H_(N) junction (in which there are 20 amino acids between the Cysresidues of the LC & H_(N) domains) and a 6 amino acid(Ile-Ser-Glu-Phe-Gly-Ser) N- terminal extension. 109 DNA coding for amaltose-binding protein-Factor Xa-intein- LC/B-Factor Xa-H_(N)expression construct. 110 MBP-LH_(N)/B produced by expression of SEQ ID109, said polypeptide incorporating a self-cleavable intein sequence tofacilitate removal of the MBP purification tag and a Factor Xa proteaseactivation site at the LC-H_(N) junction 111 DNA coding for LH_(N)/Bincorporating an enterokinase protease activation site at the LC-H_(N)junction (in which there are 11 amino acids between the Cys residues ofthe LC & H_(N) domains) and an 11 amino acid (Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser) N-terminal extension derived from the attsite adaptation of the vector. This construct has the C-terminal STOPcodon removed to facilitate direct fusion of fragment and ligands. 112LH_(N)/B produced by expression of SEQ ID 111, said polypeptideincorporating an enterokinase protease activation site at the LC-H_(N)junction (in which there are 11 amino acids between the Cys residues ofthe LC & H_(N) domains) and an 11 amino acid (Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser) N-terminal extension derived from the att siteadaptation of the vector. 113 DNA coding for LC/B with no STOP codon, alinker peptide incorporating the first 6 amino acids of the H_(N) domainand an enterokinase protease cleavage site bounded by Cys residues 114LC/B produced by expression of SEQ ID 113, said polypeptide having noSTOP codon, a linker peptide incorporating the first 6 amino acids ofthe H_(N) domain and an enterokinase protease cleavage site bounded byCys residues 115 DNA coding for LH_(N)/C incorporating a Factor Xacleavage site at the LC-H_(N) junction, an 11 amino acid (Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser) N-terminal extension derived from theatt site adaptation of the vector, and a C- terminal (Glu)₈ peptide tofacilitate molecular clamping. 116 LH_(N)/C produced by expression ofSEQ ID 115, said polypeptide incorporating a Factor Xa cleavage site atthe LC-H_(N) junction, an 11 amino acid (Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser) N-terminal extension derived from the att siteadaptation of the vector, and a C-terminal (Glu)₈ peptide to facilitatemolecular clamping. 117 DNA coding for LH_(N)/C incorporating a FactorXa cleavage site at the LC-H_(N) junction, an 11 amino acid(Thr-Ser-Leu- Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser) N-terminal extensionderived from the att site adaptation of the vector, and a C- terminalfos ligand bounded by a pair of Cys residues to facilitate molecularclamping. 118 LH_(N)/C produced by expression of SEQ ID 117, saidpolypeptide incorporating a Factor Xa cleavage site at the LC-H_(N)junction, an 11 amino acid (Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser) N-terminal extension derived from the att siteadaptation of the vector, and a C-terminal fos ligand bounded by a pairof Cys residues to facilitate molecular clamping. 119 DNA coding forLH_(N)/C incorporating a Factor Xa cleavage site at the LC-H_(N)junction, an 11 amino acid (Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser) N-terminal extension derived from theatt site adaptation of the vector, and a C- terminal (Glu)₈ peptidebounded by a pair of Cys residues to facilitate molecular clamping 120LH_(N)/C produced by expression of SEQ ID 119, said polypeptideincorporating a Factor Xa cleavage site at the LC-H_(N) junction, an 11amino acid (Thr-Ser-Leu-Tyr-Lys- Lys-Ala-Gly-Phe-Gly-Ser) N-terminalextension derived from the att site adaptation of the vector, and aC-terminal (Glu)₈ peptide bounded by a pair of Cys residues tofacilitate molecular clamping 121 DNA coding for LH_(N)/C incorporatinga Factor Xa cleavage site at the LC-H_(N) junction, an 11 amino acid(Thr-Ser-Leu- Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser) N-terminal extensionderived from the att site adaptation of the vector, and a C- terminalfos ligand to facilitate molecular clamping. 122 LH_(N)/C produced byexpression of SEQ ID 121, said polypeptide incorporating a Factor Xacleavage site at the LC-H_(N) junction, an 11 amino acid(Thr-Ser-Leu-Tyr-Lys- Lys-Ala-Gly-Phe-Gly-Ser) N-terminal extensionderived from the att site adaptation of the vector, and a C-terminal fosligand to facilitate molecular clamping 123 DNA coding for LH_(N)/Cincorporating a Factor Xa cleavage site at the LC-H_(N) junction, an 15amino acid (Ile-Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser-Leu-Asp-His) N- terminal extensionderived from the att site adaptation of the vector. 124 LH_(N)/Cproduced by expression of SEQ ID 123, said polypeptide incorporating aFactor Xa cleavage site at the LC-H_(N) junction, a 15 amino acid(Ile-Thr-Ser-Leu-Tyr-Lys- Lys-Ala-Gly-Phe-Gly-Ser-Leu-Asp-His)N-terminal extension derived from the att site adaptation of the vector.125 DNA coding for LH_(N)/C incorporating a Factor Xa cleavage site atthe LC-H_(N) junction and an 11 amino acid (Val-Pro-Glu-Phe-Gly-Ser-Ser-Arg-Val-Asp-His) N-terminal extension derivedfollowing cleavage of the protein with enterokinase 126 LH_(N)/Cproduced by expression of SEQ ID 125, said polypeptide incorporating aFactor Xa cleavage site at the LC-H_(N) junction and an11 amino acid(Val-Pro-Glu-Phe-Gly- Ser-Ser-Arg-Val-Asp-His) N-terminal extensionderived following cleavage of the protein with enterokinase to releasethe N-terminal MBP purification tag. 127 DNA coding for LH_(N)/Cincorporating a Factor Xa cleavage site at the LC-H_(N) junction and an10 amino acid (Val-Glu- Phe-Gly-Ser-Ser-Arg-Val-Asp-His) N-terminalextension derived following cleavage of the protein with genenase 128LH_(N)/C produced by expression of SEQ ID 127, said polypeptideincorporating a Factor Xa cleavage site at the LC-H_(N) junction and an10 amino acid (Val-Glu-Phe-Gly-Ser- Ser-Arg-Val-Asp-His) N-terminalextension derived following cleavage of the protein with genenase torelease the N- terminal MBP purification tag 129 DNA coding for LH_(N)/Cincorporating a Factor Xa cleavage site at the LC-H_(N) junction and an11 amino acid (Ile-Ser- Glu-Phe-Gly-Ser-Ser-Arg-Val-Asp-His) N-terminalextension derived following cleavage of the protein with Factor Xa 130LH_(N)/C produced by expression of SEQ ID 129, said polypeptideincorporating a Factor Xa cleavage site at the LC-H_(N) junction and an11 amino acid (Ile-Ser-Glu-Phe-Gly- Ser-Ser-Arg-Val-Asp-His) N-terminalextension derived following cleavage of the protein with Factor Xa 131DNA coding for LH_(N)/C incorporating a Factor Xa cleavage site at theLC-H_(N) junction, a 15 amino acid (Ile-Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser-Leu-Asp-His) N-terminal extension and a21 amino acid (Leu-Gln-Thr-Leu-Asp-Asp-Pro-Ala-Phe-Leu-Tyr-Lys-Val-Val-Ile-Phe-Gln-Asn-Ser-Asp- Pro) C-terminalextension derived from the att site adaptation of the vector. The clonehas no STOP codon in order to facilitate fusion of ligands ontoC-terminus of H_(N) domain. 132 LH_(N)/C produced by expression of SEQID 131, said polypeptide incorporating a Factor Xa cleavage site at theLC-H_(N) junction, a 15 amino acid (Ile-Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-Phe-Gly-Ser-Leu-Asp-His) N-terminal extension and a 21 aminoacid (Leu-Gln-Thr-Leu-Asp-Asp-Pro-Ala-Phe-Leu-Tyr-Lys-Val-Val-Ile-Phe-Gln-Asn-Ser-Asp- Pro) C-terminalextension derived from the att site adaptation of the vector. The clonehas no STOP codon in order to facilitate fusion of ligands ontoC-terminus of H_(N) domain. 133 DNA coding for LH_(N)/C incorporating aFactor Xa cleavage site at the LC-H_(N) junction, an N-terminalextension and a C- terminal extension derived from the att siteadaptation of the vector. The clone has no STOP codon in order tofacilitate fusion of ligands onto C-terminus of H_(N) domain. 134 DNAcoding for LC/C as prepared in pENTRY2 for use in the GATEWAY ® (cloningsystem) site specific recombination cloning system. LC/C has no STOPcodon in order to facilitate creation of LC-H_(N) fusions throughrecombination. 135 DNA coding for LH_(N)/C as prepared in pENTRY2 foruse in the GATEWAY ® (cloning system) site specific recombinationcloning system. LH_(N)/C has a STOP codon and is thus in the correctformat for recombination into an appropriate Destination VECTOR (cloningvector). 136 DNA coding for LH_(N)/C as prepared in pENTRY2 for use inthe GATEWAY ® (cloning system) site specific recombination cloningsystem. LH_(N)/C has no STOP codon in order to facilitate creation ofLH_(N)/C-ligand fusions through recombination. 137 DNA sequence of apMTL vector modified to be a suitable DESTINATION VECTOR (cloningvector) in which to insert endopeptidase fragments from ENTRY VRECTORS(cloning vectors). Vector constructed by insertion of GATEWAY ® (cloningsystem) vector conversion cassette reading frame A into pMAL-c2X.Expression cassette (ptac promoter, male gene, GATEWAY ® (cloningsystem) cassette and polylinker) subsequently cloned into pMTL. 138 DNAcoding for LH_(N)/A-ligand (Erythrina cristagalli lectin) fusion inwhich the LC-H_(N) junction incorporates a specific enterokinaseprotease activation site and the ligand is spaced from the H_(N) domainby a peptide sequence derived from an Rnase A loop 139 LH_(N)/A-ligand(Erythrina cristagalli lectin) fusion produced by expression of SEQ ID138, in which the LC-H_(N) junction incorporates a specific enterokinaseprotease activation site and the ligand is spaced from the H_(N) domainby a peptide sequence derived from an Rnase A loop 140 DNA coding fortetanus toxin 141 Tetanus toxin produced by expression of SEQ ID 140,said polypeptide incorporating the LC, H_(N) and H_(C) domains 142 DNAcoding for LH_(N) of tetanus toxin, in which the 3′ end of the cloneencodes the sequence . . . Glu-Glu-Asp-Ile-Asp- Val-STOP, terminating atresidue Val879 143 LH_(N) of tetanus toxin produced by expression of SEQID 142, said polypeptide terminating with the sequence . . . Glu-Glu-Asp-Ile-Asp-Val-STOP, terminating at residue Val879. 144 DNA coding forLH_(N) of tetanus toxin, in which the 3′ end of the clone encodes thesequence . . . Glu-Glu-Asp-Ile-Asp- Val-STOP as in SEQ ID 142. The clonealso incorporates a specific enterokinase protease activation site atthe junction of the LC and H_(N) domain. 145 LH_(N) of tetanus toxinproduced by expression of SEQ ID 144, said polypeptide terminating withthe sequence . . . Glu-Glu- Asp-Ile-Asp-Val-STOP as in SEQ ID 143. Theprotein also incorporates a specific enterokinase protease activationsite at the junction of the LC and H_(N) domain. 146 DNA coding forLH_(N) of tetanus toxin, in which the 3′ end of the clone encodes thesequence . . . Glu-Glu-Asp-Ile-Asp-Val-Ile-Leu-Lys-Lys-Ser-Thr-Ile-Leu-STOP, terminating at residue Leu887147 LH_(N) of tetanus toxin produced by expression of SEQ ID 146, saidpolypeptide terminating with the sequence . . . Glu-Glu-Asp-Ile-Asp-Val-Ile-Leu-Lys-Lys-Ser-Thr-Ile-Leu-STOP, terminating atresidue Leu887 148 DNA encoding ₂LH₄₂₃/A(Q₂E) 149 ₂LH₄₂₃/A(Q₂E), whichis a single polypeptide comprising a BoNT/A L-chain and the N-terminal423 amino acid residues of a BoNT/A H-chain. The polypeptide has beengenerated by cleavage from a GST purification tag and has a 2 amino acidextension (GS) on the N-terminus of the L- chain resulting from theproteolytic cleavage of the L-chain from the purification tag. Thepolypeptide has a variant amino acid residue E at position 2 comparedwith Q in a native serotype A L-chain. 150 DNA encoding ₂LH₄₂₃/A(Q₂E),wherein the DNA has an E. coli codon bias. 151 ₂LH₄₂₃/A(Q₂E), which isequivalent to SED ID NO 149. 152 DNA encoding LH₄₂₃/A(Q₂E), wherein theDNA has an E. coli codon bias. 153 LH₄₂₃/A(Q₂E), which is equivalent toSEQ ID NO 151 but without any N-terminal extension to the L-chain. 154DNA encoding LH₄₂₃/A(Q₂E). 155 LH₄₂₃/A(Q₂E), which is equivalent to SEQID NO 149 but without any N-terminal extension to the L-chain. 156 DNAencoding ₂L_(FXa)/₃H₄₂₃/A(Q₂E). 157 ₂L_(FXa)/₃H₄₂₃/A(Q₂E), which isequivalent to SEQ ID NO 151 and wherein a Factor Xa cleavage site hasbeen introduced between the L-chain and H-chain components of thepolypeptide. 158 DNA encoding LH₄₂₃/A(Q₂E)-6His. 159 LH₄₂₃/A(Q₂E)-6His,which is a native LH_(N) molecule and includes a C-terminal poly-Hispurification tag. 160 DNA encoding ₂L_(FXa)/₃H₄₂₃/A(Q₂E)_(Fxa)-6His. 161₂L _(FXa)/₃H₄₂₃/A(Q₂E)_(FXa)-6His, which is equivalent to SEQ ID NO 157and includes a Factor Xa cleavage site to facilitate removal of thepoly-His purification tag. 162 DNA encoding ₂LH₄₂₃/A(Q₂E, H₂₂₇Y). 163₂LH₄₂₃/A(Q₂E, H₂₂₇Y), which is equivalent to SEQ ID NO 149 and whereinthe polypeptide has a variant amino acid residue Y at position 227compared with H in a native serotype A L-chain. 164 DNA encoding₂LH₄₂₃/A(Q₂E, H₂₂₇Y), wherein the DNA has an E. coli codon bias. 165₂LH₄₂₃/A(Q₂E, H₂₂₇Y), which is equivalent to SEQ ID NO 163. 166 DNAencoding ₂LH₄₂₃/A(Q₂E, E₂₂₄Q), wherein the DNA has an E. coli codonbias. 167 ₂LH₄₂₃/A(Q₂E, E₂₂₄Q), which is equivalent to SEQ ID NO 151 andwherein the polypeptide has a variant amino acid residue Q at position224 compared with E in a native serotype A L-chain. 168 DNA encoding₂LH₄₂₃/A(Q₂E, E₂₂₄Q, H₂₂₇Y), wherein the DNA has an E. coli codon bias.169 ₂LH₄₂₃/A(Q₂E, E₂₂₄Q, H₂₂₇Y), which is equivalent to SEQ ID NO 167and wherein the polypeptide has a variant amino acid residue Y atposition 227 compared with H in a native serotype A L-chain. 170 DNAencoding L_(FXa)/H₄₁₇/B. 171 L_(Fxa)/H₄₁₇/B, which is a singlepolypeptide comprising a BoNT/B L-chain and the N-terminal 417 aminoacid residues of a BoNT/B H-chain, wherein a Factor Xa cleavage siteexists between the L-chain and H-chain. 172 DNA encoding L_(FXa)/H₄₁₇/B.173 L_(FXa)/H₄₁₇/B, which is a single polypeptide comprising a BoNT/BL-chain and the N-terminal 417 amino acid residues of a BoNT/B H-chain,wherein a Factor Xa cleavage site exists between the L-chain andH-chain. 174 DNA encoding L_(Fxa)/H₄₁₇/B. 175 L_(FXa)/H₄₁₇/B, which isequivalent to SEQ ID NO 173, wherein a modified linker sequence existsbetween the L-chain and H-chain vis-a-vis SEQ ID NO 173. 176 DNAencoding LC/A H227Y. 177 LC/A H227Y. 178 DNA encoding LH_(N)/A H227Y.179 LH_(N)/A H227Y.

EXAMPLE 1

A 2616 base pair, double stranded gene sequence (SEQ ID NO: 1) has beenassembled from a combination of synthetic, chromosomal andpolymerase-chain-reaction generated DNA (FIG. 2). The gene codes for apolypeptide of 871 amino acid residues corresponding to the entirelight-chain (LC, 448 amino acids) and 423 residues of the amino terminusof the heavy chain (H_(c)) of botulinum neurotoxin type A. Thisrecombinant product is designated the LH₄₂₃/A fragment (SEQ ID NO: 2).

Construction of the Recombinant Product

The first 918 base pairs of the recombinant gene were synthesised byconcatenation of short oligonucleotides to generate a coding sequencewith an E.coli codon bias. Both DNA strands in this region werecompletely synthesised as short overlapping oligonucleotides which werephosphorylated, annealed and ligated to generate the full syntheticregion ending with a unique Kpnl restriction site. The remainder of theLH₄₂₃/A coding sequence was PCR amplified from total chromosomal DNAfrom Clostridium botulinum and annealed to the synthetic portion of thegene.

The internal PCR amplified product sequences were then deleted andreplaced with the native, fully sequenced, regions from clones of C.botulinum chromosomal origin to generate the final gene construct. Thefinal composition is synthetic DNA (bases 1-913), polymerase amplifiedDNA (bases 914-1138 and 1976-2616) and the remainder is of C. botulinumchromosomal origin (bases 1139-1975). The assembled gene was then fullysequenced and cloned into a variety of E. coli plasmid vectors forexpression analysis.

Expression of the Recombinant Gene and Recovery of Protein Product

The DNA is expressed in E. coli as a single nucleic acid transcriptproducing a soluble single chain polypeptide of 99,951 Daltons predictedmolecular weight. The gene is currently expressed in E. coli as a fusionto the commercially available coding sequence of glutathioneS-transferase (GST) of Schistosoma japonicum but any of an extensiverange of recombinant gene expression vectors such as pEZZ18, pTrc99,pFLAG or the pMAL series may be equally effective as might expression inother prokaryotic or eukaryotic hosts such as the Gram positive bacilli,the yeast P. pastoris or in insect or mammalian cells under appropriateconditions.

Currently, E. coli harbouring the expression construct is grown inLuria-Bertani broth (L-broth pH 7.0, containing 10 g/l bacto-tryptone, 5g/l bacto-yeast extract and 10 g/l sodium chloride) at 37° C. until thecell density (biomass) has an optical absorbance of 0.4-0.6 at 600 nmand the cells are in mid-logarithmic growth phase. Expression of thegene is then induced by addition of isopropylthio-β-D-galactosidase(IPTG) to a final concentration of 0.5 mM. Recombinant gene expressionis allowed to proceed for 90 min at a reduced temperature of 25° C. Thecells are then harvested by centrifugation, are resuspended in a buffersolution containing 10 mM Na₂HPO₄, 0.5 M NaCl, 10 mM EGTA, 0.25% Tween,pH 7.0 and then frozen at −20° C. For extraction of the recombinantprotein the cells are disrupted by sonication. The cell extract is thencleared of debris by centrifugation and the cleared supernatant fluidcontaining soluble recombinant fusion protein (GST-LH₄₂₃/A) is stored at−20° C. pending purification. A proportion of recombinant material isnot released by the sonication procedure and this probably reflectsinsolubility or inclusion body formation. Currently we do not extractthis material for analysis but if desired this could be readily achievedusing methods known to those skilled in the art.

The recombinant GST-LH₄₂₃/A is purified by adsorption onto acommercially prepared affinity matrix of glutathione Sepharose andsubsequent elution with reduced glutathione. The GST affinitypurification marker is then removed by proteolytic cleavage andreabsorption to glutathione Sepharose; recombinant LH₄₂₃/A is recoveredin the non-adsorbed material.

Construct Variants

A variant of the molecule, LH₄₂₃/A (Q₂E,N₂₆K,A₂₇Y) (SEQ ID NO: 26) hasbeen produced in which three amino acid residues have been modifiedwithin the light chain of Lh₄₂₃/A producing a polypeptide containing alight chain sequence different to that of the published amino acidsequence of the light chain of BoNT/A.

Two further variants of the gene sequence that have been expressed andthe corresponding products purified are ₂₃LH₄₂₃/A (Q₂E,N₂₆K,A₂₇Y) (SEQID NO: 4) which has a 23 amino acid N-terminal extension as compared tothe predicted native L-chain of BoNT/A and ₂LH₄₂₃/A (Q₂E,N₂₆K,A₂₇Y) (SEQID NO: 6) which has a 2 amino acid N-terminal extension (FIG. 4).

In yet another variant a gene has been produced which contains a Eco 47III restriction site between nucleotides 1344 and 1345 of the genesequence given in (SEQ ID NO: 1). This modification provides arestriction site at the position in the gene representing the interfaceof the heavy and. light chains in native neurotoxin, and provides thecapability to make insertions at this point using standard restrictionenzyme methodologies known to those skilled in the art. It will also beobvious to those skilled in the art that any one of a number ofrestriction sites could be so employed, and that the Eco 47 IIIinsertion simply exemplifies this approach. Similarly, it would beobvious for one skilled in the art that insertion of a restriction sitein the manner described could be performed on any gene of the invention.The gene described, when expressed, codes for a polypeptide,L_(/4)H₄₂₃/A (SEQ ID NO: 10), which contains an additional four aminoacids between amino acids 448 and 449 of LH₄₂₃/A at a positionequivalent to the amino terminus of the heavy chain of native BoNT/A.

A variant of the gene has been expressed, L_(FXa/3)H₄₂₃/A (SEQ ID NO:12), in which a specific proteolytic cleavage site was incorporated atthe carboxy-terminal end of the light chain domain, specifically afterresidue 448 of L_(/4)H₄₂₃/A. The cleavage site incorporated was forFactor Xa protease and was coded for by modification of SEQ ID NO: 1. Itwill be apparent to one skilled in the art that a cleavage site foranother specified protease could be similarly incorporated, and that anygene sequence coding for the required cleavage site could be employed.Modification of the gene sequence in this manner to code for a definedprotease site could be performed on any gene of the invention.

Variants of L_(FXa/3)H₄₂₃/A have been constructed in which a thirddomain is present at the carboxy-terminal end of the polypeptide whichincorporates a specific binding activity into the polypeptide.

Specific examples described are:

(1) L_(FXa/3)H₄₂₃/A-IGF-1 (SEQ ID NO: 14) , in which thecarboxy-terminal domain has a sequence equivalent to that ofinsulin-like growth factor-1 (IGF-1) and is able to bind to theinsulin-like growth factor receptor with high affinity;

(2) L_(FXa/3)H₄₂₃/A-CtxA14 (SEQ ID NO: 16, in which the carboxy-terminaldomain has a sequence equivalent to that of the 14 amino acids from thecarboxy-terminus of the A-subunit of cholera toxin (CtxA) and is therebyable to interact with the cholera toxin B-subunit pentamer; and(3) L_(FXa/3)H₄₂₃/A-ZZ (SEQ ID NO: 18), in which the carboxy-terminaldomain is a tandem repeating synthetic IgG binding domain. This variantalso exemplifies another modification applicable to the currentinvention, namely the inclusion in the gene of a sequence coding for aprotease cleavage site located between the end of the clostridial heavychain sequence and the sequence coding for the binding ligand.Specifically in this example a sequence is inserted at nucleotides 2650to 2666 coding for a genenase cleavage site. Expression of this geneproduces a polypeptide which has the desired protease sensitivity at theinterface between the domain providing H_(N) function and the bindingdomain. Such a modification enables selective removal of the C-terminalbinding domain by treatment of the polypeptide with the relevantprotease.

It will be apparent that any one of a number of such binding domainscould be incorporated into the polypeptide sequences of this inventionand that the above examples are merely to exemplify the concept.Similarly, such binding domains can be incorporated into any of thepolypeptide sequences that are the basis of this invention. Further, itshould be noted that such binding domains could be incorporated at anyappropriate location within the polypeptide molecules of the invention.

Further embodiments of the invention are thus illustrated by a DNA ofthe invention further comprising a desired restriction endonuclease siteat a desired location and by a polypeptide of the invention furthercomprising a desired protease cleavage site at a desired location.

The restriction endonuclease site may be introduced so as to facilitatefurther manipulation of the DNA in manufacture of an expression vectorfor expressing a polypeptide of the invention; it may be introduced as aconsequence of a previous step in manufacture of the DNA; it may beintroduced by way of modification by insertion, substitution or deletionof a known sequence. The consequence of modification of the DNA may bethat the amino acid sequence is unchanged, or may be that the amino acidsequence is changed, for example resulting in introduction of a desiredprotease cleavage site, either way the polypeptide retains its first andsecond domains having the properties required by the invention.

FIG. 10 is a diagrammatic representation of an expression productexemplifying features described in this example. Specifically, itillustrates a single polypeptide incorporating a domain equivalent tothe light chain of botulinum neurotoxin type A and a domain equivalentto the H_(N) domain of the heavy chain of botulinum neurotoxin type Awith a N-terminal extension providing an affinity purification domain,namely GST, and a C-terminal extension providing a ligand bindingdomain, namely an IgG binding domain. The domains of the polypeptide arespatially separated by specific protease cleavage sites enablingselective enzymatic separation of domains as exemplified in the Figure.This concept is more specifically depicted in FIG. 11 where the variousprotease sensitivities are defined for the purpose of example.

Assay of Product Activity

The LC of botulinum neurotoxin type A exerts a zinc-dependentendopeptidase activity on the synaptic vesicle associated proteinSNAP-25 which it cleaves in a specific manner at a single peptide bond.The ₂LH₄₂₃/A (Q₂E,N₂₆K,A₂₇Y) (SEQ ID NO: 6) cleaves a synthetic SNAP-25substrate in vitro under the same conditions as the native toxin (FIG.3). Thus, the modification of the polypeptide sequence of ₂LH₄₂₃/A(Q₂E,N₂₆K,A₂₇Y) relative to the native sequence and within the minimalfunctional LC domains does not prevent the functional activity of the LCdomains.

This activity is dependent on proteolytic modification of therecombinant GST-₂LH₄₂₃/A (Q₂E,N₂₆K,A₂₇Y) to convert the single chainpolypeptide product to a disulphide linked dichain species. This iscurrently done using the proteolytic enzyme trypsin. The recombinantproduct (100-600 μg/ml) is incubated at 37° C. for 10-50 minutes withtrypsin (10 μg/ml) in a solution containing 140 mM NaCl, 2.7 mM KCl, 10mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.3. The reaction is terminated byaddition of a 100-fold molar excess of trypsin inhibitor. The activationby trypsin generates a disulphide linked dichain species as determinedby polyacrylamide gel electrophoresis and immunoblotting analysis usingpolyclonal anti-botulinum neurotoxin type A antiserum.

₂LH₄₂₃/A is more stable in the presence of trypsin and more active inthe in vitro peptide cleavage assay than is ₂₃LH₄₂₃/A. Both variants,however, are fully functional in the in vitro peptide cleavage assay.This demonstrates that the recombinant molecule will tolerate N-terminalamino acid extensions and this may be expanded to other chemical ororganic moieties as would be obvious to those skilled in the art.

EXAMPLE 2

As a further exemplification of this invention a number of genesequences have been assembled coding for polypeptides corresponding tothe entire light-chain and varying numbers of residues from the aminoterminal end of the heavy chain of botulinum neurotoxin type B. In thisexemplification of the disclosure the gene sequences assembled wereobtained from a combination of chromosomal and polymerase-chain-reactiongenerated DNA, and therefore have the nucleotide sequence of theequivalent regions of the natural genes, thus exemplifying the principlethat the substance of this disclosure can be based upon natural as wellas a synthetic gene sequences.

The gene sequences relating to this example were all assembled andexpressed using methodologies as detailed in Sambrook J, Fritsch E F &Maniatis T (1989) Molecular Cloning: A Laboratory Manual (2nd Edition),Ford N, Nolan C, Ferguson M & Ockler M (eds), Cold Spring HarborLaboratory Press, New York, and known to those skilled in the art.

A gene has been assembled coding for a polypeptide of 1171 amino acidscorresponding to the entire light-chain (443 amino acids) and 728residues from the amino terminus of the heavy chain of neurotoxin typeB. Expression of this gene produces a polypeptide, LH₇₂₈/B (SEQ ID NO:20), which lacks the specific neuronal binding activity of full lengthBoNT/B.

A gene has also been assembled coding for a variant polypeptide, LH₄₁₇/B(SEQ ID NO: 22), which possesses an amino acid sequence at its carboxyterminus equivalent by amino acid homology to that at thecarboxy-terminus of the heavy chain fragment in native LH_(N)/A.

A gene has also been assembled coding for a variant polypeptide, LH₁₀₇/B(SEQ ID NO: 24), which expresses at its carboxy-terminus a shortsequence from the amino terminus of the heavy chain of BoNT/B sufficientto maintain solubility of the expressed polypeptide.

Construct Variants

A variant of the coding sequence for the first 274 bases of the geneshown in SEQ ID NO: 21 has been produced which whilst being a non-nativenucleotide sequence still codes for the native polypeptide.

Two double stranded, a 268 base pair and a 951 base pair, gene sequenceshave been created using an overlapping primer PCR strategy. Thenucleotide bias of these sequences was designed to have an E.coli codonusage bias.

For the first sequence, six oligonucleotides representing the first (5′)268 nucleotides of the native sequence for botulinum toxin type B weresynthesised. For the second sequence 23 oligonucleotides representinginternal sequence nucleotides 691-1641 of the native sequence forbotulinum toxin type B were synthesised. The oligonucleotides rangedfrom 57-73 nucleotides in length. Overlapping regions, 17-20nucleotides, were designed to give melting temperatures in the range52-56° C. In addition, terminal restriction endonuclease sites of thesynthetic products were constructed to facilitate insertion of theseproducts into the exact corresponding region of the native sequence. The268 bp 5′ synthetic sequence has been incorporated into the gene shownin SEQ ID NO: 21 in place of the original first 268 bases (and is shownin SEQ ID NO: 27). Similarly the sequence could be inserted into othergenes of the examples.

Another variant sequence equivalent to nucleotides 691 to 1641 of SEQ IDNO: 21, and employing non-native codon usage whilst coding for a nativepolypeptide sequence, has been constructed using the internal syntheticsequence. This sequence (SEQ ID NO: 28) can be incorporated, alone or incombination with other variant sequences, in place of the equivalentcoding sequence in any of the genes of the example.

EXAMPLE 3

An exemplification of the utility of this invention is as a non-toxicand effective immunogen. The non-toxic nature of the recombinant, singlechain material was demonstrated by intraperitoneal administration inmice of GST-₂LH₄₂₃/A. The polypeptide was prepared and purified asdescribed above. The amount of immunoreactive material in the finalpreparation was determined by enzyme linked immunosorbent assay (ELISA)using a monoclonal antibody (BA11) reactive against a conformationdependent epitope on the native LH_(N)/A. The recombinant material wasserially diluted in phosphate buffered saline (PBS; NaCl 8 g/l, KCl 0.2g/l, Na₂HPO₄ 1.15 g/l, KH₂PO₄ 0.2 g/l, pH 7.4) and 0.5 ml volumesinjected into 3 groups of 4 mice such that each group of mice received10, 5 and 1 micrograms of material respectively. Mice were observed for4 days and no deaths were seen.

For immunisation, 20 μg of GST-₂LH₄₂₃/A in a 1.0 ml volume ofwater-in-oil emulsion (1:1 vol:vol) using Freund's complete (primaryinjections only) or Freund's incomplete adjuvant was administered intoguinea pigs via two sub-cutaneous dorsal injections. Three injections at10 day intervals were given (day 1, day 10 and day 20) and antiserumcollected on day 30. The antisera were shown by ELISA to beimmunoreactive against native botulinum neurotoxin type A and to itsderivative LH_(N)/A. Antisera which were botulinum neurotoxin reactiveat a dilution of 1:2000 were used for evaluation of neutralisingefficacy in mice. For neutralisation assays 0.1 ml of antiserum wasdiluted into 2.5 ml of gelatine phosphate buffer (GPB; Na₂HPO₄ anhydrous10 g/l, gelatin (Difco) 2 g/l, pH 6.5-6.6) containing a dilution rangefrom 0.5 μg (5×10⁻⁶ g) to 5 picograms (5×10⁻¹² g). Aliquots of 0.5 mlwere injected into mice intraperitoneally and deaths recorded over a 4day period. The results are shown in Table 3 and Table 4. It can clearlybe seen that 0.5 ml of 1:40 diluted anti-GST-₂LH₄₂₃/A antiserum canprotect mice against intraperitoneal challenge with botulinum neurotoxinin the range 5 pg-50 ng (1-10,000 mouse LD50; 1 mouse LD50=5 pg).

TABLE 3 Neutralisation of botulinum neurotoxin in mice by guinea piganti-GST-₂LH₄₂₃/A antiserum. Botulinum Toxin/mouse Survivors Control OnDay 0.5 μg 0.005 μg 0.0005 μg 0.5 ng 0.005 ng 5 pg (no toxin) 1 0 4 4 44 4 4 2 — 4 4 4 4 4 4 3 — 4 4 4 4 4 4 4 — 4 4 4 4 4 4

TABLE 4 Neutralisation of botulinum neurotoxin in mice by non-immuneguinea pig antiserum. Botulinum Toxin/mouse Survivors Control On Day 0.5μg 0.005 μg 0.0005 μg 0.5 ng 0.005 ng 5 pg (no toxin) 1 0 0 0 0 0 2 4 2— — — — — 0 4 3 — — — — — — 4 4 — — — — — — 4

EXAMPLE 4 Expression of Recombinant LH₁₀₇/B in E. coli

As an exemplification of the expression of a nucleic acid coding for aLH_(N) of a clostridial neurotoxin of a serotype other than botulinumneurotoxin type A, the nucleic acid sequence (SEQ ID NO: 23) codingfor-the polypeptide LH₁₀₇/B (SEQ ID NO: 24) was inserted into thecommercially available plasmid pET28a (Novogen, Madison, Wis., USA). Thenucleic acid was expressed in E. coli BL21 (DE3) (New England BioLabs,Beverley, Mass., USA) as a fusion protein with a N-terminal T7 fusionpeptide, under IPTG induction at 1 mM for 90 minutes at 37° C. Cultureswere harvested and recombinant protein extracted as described previouslyfor LH₄₂₃/A.

Recombinant protein was recovered and purified from bacterial pastelysates by immunoaffinity adsorption to an immobilised anti-T7 peptidemonoclonal antibody using a T7 tag purification kit (New EnglandbioLabs, Beverley, Mass., USA). Purified recombinant protein wasanalysed by gradient (4-20%) denaturing SDS-polyacrylamide gelelectrophoresis (Novex, San Diego, Calif., USA) and western blottingusing polyclonal anti-botulinum neurotoxin type antiserum or anti-T7antiserum. Westem blotting reagents were from Novex, immunostainedproteins were visualised using the Enhanced Chemi-Luminescence system(ECL) from Amersham. The expression of an anti-T7 antibody andanti-botulinum neurotoxin type B antiserum reactive recombinant productis demonstrated in FIG. 13.

The recombinant product was soluble and retained that part of the lightchain responsible for endopeptidase activity.

The invention thus provides recombinant polypeptides useful inter aliaas immunogens, enzyme standards and components for synthesis ofmolecules as described in WO-A-94/21300.

EXAMPLE 5 Expression and Purification of LH_(N)C

The LH_(N)C DNA fragment from the native clostridial neurotoxin gene wassubcloned as a SalI-PstI fragment into the expression vector pMal-c2x(New England Biolabs). The gene fragment and the protein product thatwould be produced after proteolytic processing from the MBP-fusionprotein are defined in SEQ ID 129/130. Other commercially availableexpression systems such as pET vector (Novagen) pGEX vectors (Pharmacia)or pQE vectors (Qiagen) would also be suitable for expression of thegene fragments.

The expression clone was transferred into the host strain AD494(Novagen) containing a pACYC plasmid carrying the tRNA genes for thecodons ATA, AGA, and CTA (commercially available, for example, asRosetta strains from Novagen). As these codons are rarely used inE.coli, but are frequent in the clostridial genes encoding neurotoxins,the inclusion of these tRNA genes significantly increases expressionlevels. Those familiar with the art would recognise that this effect isnot limited to LH_(N)/C but is broadly applicable to all nativeclostridial LH_(N) fragments. Similar effects were observed in otherhost strains including HMS174 (Novagen) and TB1 (NEB), and a wide rangeof other hosts would be suitable for expression of these fragments.

Expression cultures of AD494 (PACYC tRNAs) pMalc2x LH_(N)/C were grownin Terrific Broth containing 35 μg/ml chloramphenicol, 100 μg/mlampicillin, 1 μM ZnCl₂ and 0.5% (w/v) glucose with an overnight culturediluted 1:100 into fresh media and grown for approximately 3 hours at37° C. to an OD₆₀₀ of 0.6-1. The cultures were induced with 1 mM IPTGand grown at 30° C. for 3-4 hours. Other expression systems used similarconditions except that the antibiotic was changed to kanamycin. Cellswere lysed by either sonication in column buffer (20 mM Hepes 125 mMNaCl 1 μM ZnCl₂ pH 7.2) or suitable detergent treatment (e.g. Bugbusterreagent; Novagen) and cell debris pelleted by centrifugation.Supernatant proteins were loaded onto an amylose resin columnequilibrated in column buffer and proteins eluted with a single stepelution using column buffer with 10 mM maltose.

The MBP-LH_(N)/C construct used in this example has a factor Xa sitesituated between the MBP and LH_(N) domains and also has a factor Xasite between the L and H_(N) domains to allow the formation of thedi-chain LH_(N) form. To remove the fusion tag and in this case toactivate the LH_(N) fragment, the eluted protein from the amylose columnis treated with factor Xa at a concentration of 1 unit protease activityper 50 μg purified fusion protein (as outlined by the manufacturer e.g.NEB) for approximately 20 hours at 25° C. The protein is then diluted1:5 with 20 mM Hepes pH 7.2 and loaded onto a Q-sepharose fast flowcolumn, the column washed and proteins eluted using a linear gradient of25-500 mM NaCl in the 20 mM Hepes buffer. The free LH_(N) fragment iseluted at approximately 50 mM NaCl with uncleaved fusion protein andfree MBP eluted at higher concentrations of NaCl.

Those familiar with the art will recognise that for alternativeexpression vectors such as pMal-c2g, where the site for removal of theMBP tag is genenase, two subsequent protease cleavage reactions would berequired for removal of the fusion partner (genenase cleavage) andsubsequent activation of the LH_(N) (factor Xa digestion). Thesecleavage reactions could be carried out simultaneously or with anintermediate ion exchange purification to remove contaminating proteins.An example of this model of purification/activation is identified below.These considerations are equally valid for native or syntheticactivation sites as detailed in the sequence information and for LH_(N)fragments of all the serotypes.

EXAMPLE 6 Expression and Purification of LH_(N)/F

The LH_(N) fragment from the native BoNT/F gene was modified by PCR toincorporate BamHI and HindIII, or other suitable sites, at the 5′ and 3′ends respectively. The gene fragment was cloned into pET 28 to maintainthe reading frames with the N-terminal His₆ purification tag. Theexpression clone was transferred to a host strain carrying the pACYCtRNA plasmid as outlined in example 5 and the DE3 lysogen carrying theT7 polymerase gene. Suitable host strains would include JM109, AD494,HMS174, TB1 TG1 or BL21 carrying the appropriate genetic elements. Forexample HMS174 (DE3) pACYC tRNA pET28a LH_(N)/F was used for expressionand purification.

Expression cultures of HMS174 (DE3) PACYC tRNA pET28a LH_(N)/F weregrown in Terrific Broth containing 35 μg/ml chloramphenicol, 35 μg/mlkanamycin, 1 μM ZnCl₂ and 0.5% (w/v) glucose to an OD₆₀₀ of 2.0 at 30°C. and cultures were induced with 500 μM IPTG and grown at 25° C. for 2hours prior to harvest by centrifugation. The cells were lysed in 20 mMHepes 500 mM NaCl pH 7.4 by sonication or detergent Iysis and thesoluble protein fraction loaded onto a metal chelate column (e.g. IMACHiTrap column Amersham-Pharmacia) loaded with CuSO₄. Protein was elutedusing a linear gradient of imidazole with His₆ LH_(N)/F eluting atbetween 50 and 250 mM imidazole.

The His₆ tag was removed by treatment with thrombin essentially asdescribed in Example 5. The released LH_(N) fragment was purified usingion exchange on a Q-sepharose column as described in Example 5.

EXAMPLE 7 Expression and Purification of LH_(N)TeNT

A native LH_(N)TeNT gene fragment was modified to replace the nativelinker region with an enterokinase cleavable linker as shown in SEQ ID144/145 and to incorporate cloning sites at the 5′ (BamHI ) and 3′ ends(HindIII). This fragment was subcloned into pMAL c2x and expressed inHMS174 (PACYC tRNA) as described in Example 5. Initial purification onan amylose resin column, cleavage with factor Xa to remove the fusiontag and the ion exchange purification was also as described in Example 5except that the positions of the elution peaks were reversed with thefree MBP peak eluting before the peak for free LH_(N).

EXAMPLE 8 Expression of LH_(N)/C from a GATEWAY® (Cloning System)Adapted Expression Vector

The LH_(N)C fragment was cloned into a GATEWAY® (cloning system) ENTRYVECTOR (cloning vector) as a SalI-PstI. Two version were made with astop codon within the 3′ PstI site to terminate the protein at thisposition (LH_(N)C STOP; SEQ ID 123/124), or with no stop codon to allowthe expression of the fragment with C-terminal fusion partners (LH_(N)CNS; SEQ ID 131/132). The ENTRY VECTOR (cloning vector) was recombinedwith the DESTINATION VECTOR (cloning vector) to allow expression of thefragment with an N-terminal MBP tag. Recombination was according tostandard protocols (Invitrogen GATEWAY® (cloning system) expressionmanual).

Expression of the fusion protein from the strain AD494 (PACYC tRNA)pMTL-malE-GW LH_(N)C STOP, and its purification and was as described inExample 5. The addition of the additional N-terminal sequence made nosignificant change to the overall expression and purification. The finalproduct following factor Xa cleavage was a disulfide bonded di-chainfragment as described above.

For expression of the fragment with additional C-terminal domains theLH_(N)C NS ENTRY VECTOR (cloning vector) was recombined with aDESTINATION VECTOR (cloning vector) carrying additional sequencesfollowing the attachment site and in the appropriate frame. The sequenceof the DNA encoding the LH_(N)/C fragment flanked by att sites that hasthe properties necessary to facilitate recombination to create a fullfusion is described in SEQ ID 133. For example, the DESTINATION VECTOR(cloning vector) pMTL-malE-GW-att-IGF was produced by subcloning thecoding sequence for human IGF as an XbaI-HindIII fragment into theappropriate sites. Recombination of the LH_(N)/C NS fragment into thisvector yielded pMTL-malE-GW-LH_(N)C-att-IGF.

This clone was expressed and purified as described above. Additionalpurification methods utilising the binding properties of the C-terminalIGF domain could also be used if desired.

Those familiar with the art will recognise that a similar approach couldbe used for other LH_(N) fragments from either BoNT/C or otherserotypes. Similarly other C-terminal purification tags or ligands couldbe incorporated into DESTINATION VECTORs (cloning vectors) in the sameway as for IGF above.

EXAMPLE 9 Expression of LH_(N)TeNT from a GATEWAY® (Cloning System)Adapted Expression Vector

The LH_(N)TeNT BamHI-HindIII fragment described in Example 7 wassubcloned into an ENTRY VECTOR (cloning vector) to maintain theappropriate reading frames. The ENTRY VECTOR (cloning vector) wasdesigned to incorporate a factor Xa site immediately adjacent to theBamHI site such that cleavage resulted in a protein starting with theGlySer residues encoded by the BamHI site. The ENTRY VECTOR (cloningvector) was recombined with a commercially available DESTINATION VECTOR(cloning vector) carrying an N-terminal 6-His tag (e.g. pDEST17;Invitrogen). The resulting clone pDEST17 LH_(N)TeNT was expressed in thehost strain HMS174 (pACYC tRNA). As described in Example 6. Purificationof the fusion protein is also as described in Example 5 with theN-terminal His tag removed by factor Xa treatment, followed bysubsequent removal of factor Xa on a Q-sepharose column.

EXAMPLE 10 Directed Coupling of an LH_(N)/B Fragment and a Ligand via aFos/Jun or Glu/Arg Molecular Clamp

LH_(N)/C clones of the type described in SEQ ID 115/116, 117/118,119/120& 121/122 were expressed and purified as previously indicated in Example5. Purified, activated LH_(N)/C protein was then mixed with an equimolaramount of ligand tagged with the complementary clamp partner gun-taggedligand for SEQ ID 117/118 and 121/122; poly-arginine-tagged ligand forSEQ ID 115/116 and 119/120). Proteins were gently mixed to facilitateassociated, then purified to isolate associated ligand-endopeptidasefragment.

EXAMPLE 11 Directed Coupling of an LH_(N)TeNT Fragment and a Ligand viaan Acid/Base Molecular Clamp

LH_(N)TeNT clones of the type described in SEQ ID 142/143,144/145 &146/147 were modified to incorporate one component of the acid/baseleucine zipper clamping system. Following expression and purification ofthe tagged proteins as previously indicated in Example 5, theassociation with tagged ligand was performed essentially as described inExample 10.

EXAMPLE 12 Activation of LH_(N)/B, Carrying a Thrombin ProteaseProcessing Site, to Yield a Di-chain Fragment

As in SEQ ID 99/100 an LH_(N)/B carrying a thrombin site in the linkerbetween the L and H_(N) domains was expressed from pMAL c2x essentiallyas described in Example 5. The purified LH_(N)/B fragment was incubatedwith 1 unit thrombin per mg protein for 20 hours at 25° C. The di-chainLH_(N) was separated form the thrombin by further purification on aQ-sepharose column as described in Example 5

EXAMPLE 13 Activation of LH_(N)TeNT Carrying an Enterokinase ProcessingSite to Yield a Di-chain Fragment

To prepare activated di-chain LH_(N) the purified protein (e.g. obtainedfrom SEQ ID 144/145) was treated with enterokinase at a concentration of1 enzyme unit per 50 μg purified protein at 25° C. for 20 hours. Theactivated di-chain LH_(N) was then purified from the enterokinase by ionexchange on a Q-sepharose column under identical conditions to that usedfor the purification following factor Xa cleavage (as described inExample 5) or using a benzamidine sepharose column equilibrated in 20 mMHepes 100 mM NaCl pH7.2 to specifically bind and remove theenterokinase.

EXAMPLE 14 Methodologies for Assessment of Vaccine Candidates

A variety of methodologies exist that may be implemented to assesscandidate vaccines based on clostridial neurotoxins. They are generallyseparated into two types of assay: the first being a protection assayinvolving direct challenge of previously ‘vaccinated’ animals with astandard quantity of BoNT; the second being an assessment of theneutralising capability of the antisera obtained in response to exposureof the animal to the vaccine candidate.

Protection Assay

For the protection assay, groups of 10 mice (16-22 g) are used. Mice areinoculated three times at 0, 2 and 4 weeks, with 5 or 15 μg of immunogen(100 μl per mouse). Mice are challenged 2 weeks after the finalvaccination with 1000 or 10000 mouse LD₅₀ of BoNT/A and observed for 4days following challenge. For controls, groups of naive mice arechallenged with the same levels of toxin.

Neutralisation Assay

Based on US Army Biological Laboratories Technical Study 46 (Cardellaand Wright, 1964), this test provides an accurate titration of theneutralising immune response in guinea pigs which can be calibratedusing antiserum of known potency (IU). For this protocol, groups of 4guinea-pigs are given 1 ml of absorbed vaccine or vaccine test materialcontaining various vaccine doses. After 30 days, guinea pigs are bledand serum pooled using equal volumes from each animal. Once guinea-pigantiserum has been generated, the toxin neutralising activity can beassessed by two methods:

(i) Mouse Neutralisation Assay

In this assay a fixed concentration of neurotoxin (usually about 10³mouse LD₅₀) is mixed with serial dilutions of antiserum, incubated for 1hour and then injected into groups of mice. Comparison of the antiserumdilution titre, which is fully protective, to that of a control orstandard antiserum allows the titre of the test antiserum to bedetermined.

Alternatively, for a less quantitative assessment of neutralisingefficacy, the antisera concentration is fixed at 10-100 μl and mixedwith varying mouse LD₅₀ of toxin for 1 hour prior to injection intomice.

(ii) In Vitro Assay using Embryonic Spinal Cord Neurons

The inhibitory effects of the BoNTs on the release of glycine from eSNscan also be blocked in a dose-dependent manner by pre-mixing the toxinwith increasing amounts of antiserum (Hall et al., 2004). The assaysmeasures the stimulated release of glycine using a protocol previouslydescribed (Duggan et al., 2002, J. Biol. Chem., 277, 34846-34852). Sincethe eSN assay is a close mimic to the effects of toxin at theneuromuscular junction at the mechanistic level, it is a useful assayfor measuring the neutralizing activity of antisera. A fixedconcentration of toxin is mixed with various dilutions of the testantiserum. Titres are compared with standard antiserum of known titre.

EXAMPLE 15 Assessment of the Protective Efficacy of Anti-LH_(N) Antiseraby In Vivo Analysis

Using the neutralisation assay described in Example 14, the neutralisingeffects of rabbit α-LH_(N)/A antisera generated using non-toxoidedrecLH_(N)/A were assessed. It was found that pre-incubation ofanti-LH_(N)/A antisera with up to 3.2×10⁴ LD₅₀ BoNT prevented all animaldeaths. In contrast, incubation of a similar quantity of pre-immuneserum with only 1.6×10¹ LD₅₀ BoNT/A resulted in no surviving animals.Therefore, antisera raised to the LC and H_(N) domains was effective atinhibiting BoNT/A holotoxin-mediated toxic effects.

EXAMPLE 16 Assessment of the Protective Efficacy of Anti-LH_(N) Antiseraby In Vitro Analysis

Using the in vitro neuron-based neutralisation assay described inExample 14, in vitro antibody assessment data confirmed thatanti-LH_(N)/A antisera had BoNT/A neutralising ability (see FIG. 16).BoNT/A was applied at 3 pM (450 pg/ml) to achieve approximately 50%inhibition of [³H] glycine release. Purified antisera was mixed inincreasing excesses to the toxin, incubated for 1 hour at 37° C., andapplied to eSCNs in multi-well plates. Control antisera ((c);non-specific rabbit IgG) was used to control for non-specific effects.Cultures were incubated at 37° C., 10% CO₂ for 16 hours beforeassessment of stimulated [³H] glycine release. % Inhibition wascalculated relative to net release from non-treated controls. Rbα-LH_(N)/A fully inhibited the effects of 3 pM BoNT/A when mixed at a10000-molar excess of antibody.

In addition, the ability of anti-recLH_(N)/A antisera to neutralise theeffects of BoNT/A, B and C, (3, 50 and 50 pM respectively) was assessedusing the eSCN model (see FIG. 17). Purified antisera was mixed witheach of the toxins, incubated for 1 hour at 37° C., and applied to eSCNsin multi-well plates. Cultures were incubated at 37° C., 10% CO₂ for 16hours before assessment of stimulated [³H] glycine release. % Inhibitionwas calculated relative to net release from non-treated controls. Dataare representative of 3 determinations ±SEM. No inhibitory effect wasseen for BoNT/B & BoNT/C, (at similar antibody:toxin excesses as usedfor BoNT/A) suggesting that the protective effect of the antibody wasserotype specific.

EXAMPLE 17 Assessment of the Ability of Anti-LH_(N) Antisera to Inhibitthe Action of Retargeted Clostridial Endopeptidases

Using the in vitro neuron-based neutralisation assay described inExample 14, in vitro antibody assessment data demonstrated thatanti-LH_(N)/A antisera were also able to inhibit the biological effectsof non-H_(C) mediated entry of the LC into target cells.

Wheatgerm agglutinin-LH_(N)/A conjugate was prepared as described inChaddock et al., 2000, Infection & Immunity 68, 2587-2893. The abilityof anti-recLH_(N)/A antisera (A) and anti-H_(C) antisera (B) toneutralise the effects of 1 μg/ml WGA-LH_(N)/A was assessed using theeSCN model (see FIGS. 18A and B). Purified antisera was mixed inincreasing molar excesses to the conjugate, incubated for 1 hour at 37°C., and applied to eSCNs in multi-well plates. Control antisera ((c);non-specific rabbit IgG) was used to control for non-specific effects.Cultures were incubated at 37° C., 10% CO₂ for 16 hours beforeassessment of stimulated [³H] glycine release. % Inhibition wascalculated relative to net release from non-treated controls. Data arerepresentative of 3 experiments ±SEM. FIG. 18A shows inhibition of awheat-germ agglutinin-LH_(N)/A conjugate effect using anti-LH_(N)/Aantisera. By contrast, the data described in FIG. 18B shows thatantisera raised to the H_(C) domain were ineffective at inhibiting theactions of the wheat-germ agglutinin-LH_(N)/A conjugate.

EXAMPLE 18 Assessment of the Direct Immunisation Protective Capabilityof LH_(N)

Using the protection assay methodology described in Example 14, micewere inoculated with 5 μg or 15 μg LC/A, and were found to be 100%susceptible to challenge with 10³ or 10⁴ MLD50 BoNT/A. However, micethat were inoculated with 5 μg or 15 μg LH_(N)/A were 100% protected tochallenge with 10³ or 10⁴ MLD50 BoNT/A (see Table 5).

TABLE 5 Geometric Inoculation mean ELISA Challenge Survivors/ TestProtein dose titre dose total mice LH_(N)/A  5 μg 11.1 × 10⁵ 10³ MLD₅₀10/10 LH_(N)/A  5 μg 11.1 × 10⁵ 10⁴ MLD₅₀ 10/10 LH_(N)/A 15 μg 3.25 ×10⁵ 10³ MLD₅₀ 10/10 LH_(N)/A 15 μg 3.25 × 10⁵ 10³ MLD₅₀ 10/10 Naïve mice 0 <100 10³ MLD₅₀  0/10 Naïve mice  0 <100 10⁴ MLD₅₀  0/10

EXAMPLE 19 Assessment of the Protective Capability of LH_(N)/A andLH_(N)/B

Using a form of the neutralisation assay described in Example 14, theefficacy of LH_(N)/A and LH_(N)/B was assessed.

Groups of 4 guinea pigs (300-350 g) were given 1 ml of absorbed vaccinetest material containing 20 μg of vaccine at day 0 and day 14. At day 28the guinea pigs are bled and serum pooled using equal volumes from eachanimal. Once guinea-pig antiserum has been generated, the toxinneutralising activity is assessed by the mouse neutralisation test.Briefly, 2 ml of a fixed concentration of neurotoxin (10³ mouse LD₅₀) ismixed with 2 ml of a dilution of antiserum, incubated for 2 hours andthen 0.5 ml injected into each of 4 mice. In this study, the dilutionsof antisera assessed were 1:2.5, 1:5, 1:10, 1:20, 1:40, 1:80, 1:160,1:320 and 1:640. The mice were observed for 4 days and number ofsurvivors recorded.

Comparison of the antiserum dilution titre, which is fully protective,to that of a control or standard antiserum allows the titre of the testantiserum to be determined. The dilution at which the antisera providedfull protection is recorded in Table 6. Noting that a 50 IU/mlInternational standard antiserum protects at a dilution of 1:1200enables the approximate IU/ml for the guinea pig antisera to becalculated. These data are recorded in Table 7. The data illustrate thatin both cases the LH_(N) fragment was more effective at eliciting aprotective response than the equivalent toxoid material.

TABLE 6 Dilution of guinea pig antisera to fully protect mice against achallenge of 10³ MLD₅₀ BoNT Toxoid- Toxoid- LH_(N)/A LH_(N)/B BoNT/ABoNT/B Experiment 1 135 45 45 5 Experiment 2 80 40 40 5 Mean 107 42 42 5

TABLE 7 Calculated IU/ml for guinea pig antisera Toxoid- Toxoid-LH_(N)/A LH_(N)/B BoNT/A BoNT/B Experiment 1 5.6 1.9 1.9 0.21 Experiment2 3.3 1.7 1.7 0.21 Mean 4.5 1.8 1.8 0.21

EXAMPLE 20 Formulation of a Vaccine Composition Comprising a Polypeptideof the Invention

In order to achieve successful immune response to a polypeptide of theinvention, it is necessary to create a formulation in which thepolypeptide is delivered to the recipient within an appropriate carriermatrix. The carrier matrix of choice for botulinum neurotoxinfragment-based vaccines has traditionally included aluminium hydroxideas the adsorbing agent, though other alternatives (for example aluminiumphosphate) do exist.

To prepare an appropriate formulation, the LH_(N) vaccine candidates areadsorbed to aluminium hydroxide at a variety of antigen concentrationsin order to select the most appropriate concentration for maximumefficacy. Commercially available GMP aluminium hydroxide (Alhydrogel™)is used. The primary parameters that are evaluated when optimising theformulation are pH, buffer and aluminium adjuvant /antigen ratios.Buffers containing phosphate or nitrogen are avoided as phosphate isknown to impact adjuvant stability and nitrogen interferes with thedetermination of bound total protein nitrogen.

Using the simple approach of mixing commercially available Alhydrogel(Biosector 1.3) with LH_(N)/A or LH_(N)/B, singly or in combination,then assessing the absorbance of the supernatant (i.e. the non-adsorbedprotein) the binding efficacy of the LH_(N) to aluminium hydroxide isdetermined.

In a specific embodiment of the invention, LH_(N)/A or LH_(N)/B (to amaximum concentration of 0.3 mg/ml final) were mixed with alhydrogel(0.15% (v/v), equilibrated against 5 mM Hepes pH 7.4+ either 0.05 M or0.1 M NaCl) for 1 hr at room temperature with occasional agitation.After this time, the mixture was centrifuged and the supernatant OD 280nm read. The results suggest complete absorption of both LH_(N)/A and Bunder the same conditions.

Absorbance at 280 nm Alhydrogel + 0.05M Alhydrogel + 0.1M Control NaClNaCl LH_(N)/A 0.2 0.01 0.013 (94%) LH_(N)/B 0.15 0.01  0.01 (93%)

EXAMPLE 21 Formulation of a DNA Vaccine Composition

DNA vaccines are formulated by any of the following methods:

-   -   1. Lyophilisation in simple buffer +/− additional carrier        chemicals (e.g. trehalose, polyvinyl pyrrolidone (PVP) or        polyethylene glycol (PEG)). Ref DNA Vaccines: Methods and        Protocols, edited by D. B. Lowrie & R. G. Whalen, Humana Press,        2000, p 23-34, the content of which are incorporated into this        specification in their entirety by reference.    -   2. Addition of chemical adjuvants. Ref DNA Vaccines: Methods and        Protocols, edited by D. B. Lowrie & R. G. Whalen, Humana Press,        2000, p 241-249, the content of which are incorporated into this        specification in their entirety by reference. Standard chemical        adjuvants (such as aluminium hydroxide, calcium phosphate, or        cholera toxin) have all had success in enhancing the immune        response to DNA vaccines.    -   3. Addition of genetic adjuvants. Ref DNA Vaccines: Methods and        Protocols, edited by D. B. Lowrie & R. G. Whalen, Humana Press,        2000, p 251-260, the content of which are incorporated into this        specification in their entirety by reference. Vaccine encoding        DNA and cytokine-encoding DNA are co-introduced.    -   4. Use of liposomes. Ref DNA Vaccines: Methods and Protocols,        edited by D. B. Lowrie & R. G. Whalen, Humana Press, 2000, p        305-311, the content of which are incorporated into this        specification in their entirety by reference.    -   5. Encapsulation into microparticles. See U.S. Pat Nos.        6,743,444 and 6,667,294. Methods contained within U.S. Pat. No.        6,743,444 (“Method of making microencapsulated DNA for        vaccination and gene therapy”) are directly relevant to the        preparation of DNA vaccines of the type described herein.

EXAMPLE 22 Formulation of a Composition Comprising a Passive TherapyAgent

One preferred approach to obtain antisera suitable for use in passiveimmunotherapy in humans is to use human IgG. In order to obtain humanimmune globulin, a donor (ideally more than one) is identified and,following vaccination of the donor with the composition, immune globulinis obtained from the donor using standard plasmaphoresis methods. Theimmune globulin is purified using standard methods, such as Cohncold-ethanol fractionation, or standard chromatography methods, such assizing column chromatography or antibody affinity chromatography (e.g.,using Protein A). Up to two times per week, whole blood (500 ml-1 L) isobtained from donors, plasma is isolated by centrifugation, and cellsare returned to the donors. Preferably, the purified sample contains allor predominantly IgG, but mixtures containing, e.g., IgG, IgA, and IgM,can also be used in the invention.

The botulinum endopeptidase immune globulin, prepared as describedabove, can be percutaneously (e.g., intramuscularly, intravenously, orintraperitoneally) administered to patients that have, or are at risk ofdeveloping, Clostridium botulinum infection. The C. botulinum immuneglobulin is administered in amounts ranging from 100 μg/kg-100 mg/kg, or1-50 mg/kg, for example, about 15 mg/kg, depending on donor titre: thehigher the neutralization titre of the immune globulin, the lower theamount is that needs to be administered. The immune globulin isadministered in, e.g., one or more doses. For example, in the case oftherapeutic passive immunization, an initial dose is administered fortreatment and a second dose is administered to prevent relapse.

Similar procedures have been utilised for the passive therapy of C.difficile infection (U.S. Pat. No. 6,680,168).

REFERENCE TO A SEQUENCE LISTING APPENDIX SUBMITTED ON A COMPACT DISC

The Sequence Listing written in file “Sequence Listing.txt,” 1.21megabytes, created on Jun. 6, 2005 on two identical copies of compactdiscs for application Ser. No. 11/077,550, Shone et al., RecombinantToxin Fragments, is herein incorporated-by-reference.

1. An antigenic composition comprising a single chain polypeptidecomprising first and second domains, wherein: said first domain is aclostridial neurotoxin light chain or a variant thereof, or a fragmentof said light chain or variant wherein said variant or fragment has acommon antigenic cross-reactivity to said clostridial neurotoxin lightchain; and said second domain is a clostridial neurotoxin heavy chainH_(N) portion or a variant thereof, or a fragment of said heavy chainH_(N) portion or variant wherein said variant or fragment has a commonantigenic cross-reactivity to said clostridial neurotoxin heavy chainH_(N) portion; and wherein said second domain is capable of (i)translocating the polypeptide into a cell or (ii) increasing thesolubility of the polypeptide compared to the solubility of the firstdomain on its own or (iii) both translocating the polypeptide into acell and increasing the solubility of the polypeptide compared to thesolubility of the first domain on its own; and wherein the second domainlacks a functional C-terminal part of a clostridial neurotoxin heavychain designated H_(C) thereby rendering the polypeptide incapable ofbinding to cell surface receptors that are the natural cell surfacereceptors to which native clostridial neurotoxin binds.
 2. An antigeniccomposition according to claim 1, wherein said clostridial neurotoxinlight chain is a botulinum neurotoxin light chain.
 3. An antigeniccomposition according to claim 1, wherein said clostridial toxin lightchain is a tetanus neurotoxin light chain.
 4. An antigenic compositionaccording to claim 1, wherein said clostridial neurotoxin heavy chain isa botulinum neurotoxin heavy chain.
 5. An antigenic compositionaccording to claim 1, wherein said clostridial toxin heavy chain is atetanus neurotoxin heavy chain.
 6. An antigenic composition according toclaim 1, wherein said second domain is a clostridial neurotoxin heavychain H_(N) portion.
 7. An antigenic composition according to claim 1,wherein the second domain lacks a C-terminal part of a clostridialneurotoxin heavy chain designated H_(C), thereby rendering thepolypeptide incapable of binding to cell surface receptors that are thenatural cell surface receptors to which native clostridial neurotoxinbinds.
 8. An antigenic composition according to claim 1, wherein one orboth of said clostridial neurotoxin light chain and said clostridialneurotoxin heavy chain is a botulinum neurotoxin type A chain.
 9. Anantigenic composition according to claim 8, wherein the botulinum toxintype A light chain has at residue 2 a glutamate, at residue 26 a lysineand at residue 27 a tyrosine.
 10. An antigenic composition according toclaim 1, wherein the second domain comprises the 423 N-terminal aminoacids of botulinum toxin type A heavy chain.
 11. An antigeniccomposition according to claim 1, wherein one or both of saidclostridial neurotoxin light chain and said clostridial neurotoxin heavychain is a botulinum neurotoxin type B chain.
 12. An antigeniccomposition according to claim 1, wherein the second domain comprisesthe 107 N-terminal amino acids of a botulinum toxin type B heavy chain.13. An antigenic composition according to claim 1, wherein the seconddomain comprises the 417 N-terminal amino acids of botulinum toxin typeB heavy chain.
 14. An antigenic composition according to claim 1,wherein the clostridial neurotoxin light chain is a botulinum toxin typeB light chain, and the second domain comprises the 417 N-terminal aminoacids of a botulinum toxin type B heavy chain.
 15. An antigeniccomposition according to claim 1, wherein one or both of saidclostridial neurotoxin light chain and said clostridial neurotoxin heavychain is a tetanus toxin chain.
 16. An antigenic composition accordingto claim 1, wherein the second domain comprises the 422 N-terminal aminoacids of tetanus heavy chain.
 17. An antigenic composition according toclaim 1, wherein the second domain comprises the 100 N-terminal aminoacids of a clostridial neurotoxin heavy chain.
 18. An antigeniccomposition according to claim 16 lacking a portion designated H_(C) ofa clostridial neurotoxin heavy chain.
 19. An antigenic compositionaccording to claim 17 lacking a portion designated H_(C) of aclostridial neurotoxin heavy chain.
 20. An antigenic compositionaccording to claim 1 comprising a site for cleavage by a proteolyticenzyme.
 21. An antigenic composition according to claim 20, wherein thecleavage site is not present in a native clostridial neurotoxin.
 22. Anantigenic composition according to claim 20, wherein the site forcleavage allows proteolytic cleavage of the first and second domains.23. An antigenic composition according to claim 20 produced by a processcomprising (a) inserting a first nucleic acid sequence encoding saidcleavage site into a second nucleic acid sequence encoding the singlechain polypeptide according to claim 1 and (b) expressing said first andsecond nucleic sequences to obtain said single chain polypeptide. 24.The antigenic composition of claim 1, wherein said single chainpolypeptide is selected from the group consisting of: SEQ ID Nos. [2, 4,6, 10, 12, 16, 20, 22, 24, 26, 30, 32, 34, 36, 38, 60, 62, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86,] 88, [90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114,116, 118, 120, 122,] 124, [126, 128, 130, 132,]143, [145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,] 169,[171, 173, 175, 177] and
 179. 25. An antigenic composition comprising asingle chain polypeptide encoded by a nucleic acid selected from thegroup consisting of SEQ ID Nos. [1, 3, 5, 9, 11, 13, 15, 19, 21, 23, 25,27, 28, 29, 31, 33, 35, 37, 59, 61, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85,] 87, [89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121,] 123, [125, 127, 129, 131, 133, 135, 136,] 142,[144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,] 168, [170, 172,174, 176,] and
 178. 26. A method of immunising against clostridialneurotoxin poisoning in a subject comprising administering to saidsubject a therapeutically effective amount of a composition according toclaim
 1. 27. An antigenic composition according to claim 1, wherein saidvariant of a light chain, or said fragment of said light chain orvariant, has a common antigenic cross-reactivity to said light chain inan assay selected from: 1) a protection assay involving direct challengeof previously vaccinated animals with clostridial neurotoxin; and 2) anassay of the neutralising capability of the anti-sera obtained inresponse to exposure of an animal to said antigenic composition.
 28. Anantigenic composition according to claim 1, wherein said variant of aheavy chain H_(N) portion, or said fragment of said heavy chain H_(N)portion or variant, has a common antigenic cross-reactivity to saidheavy chain H_(N) portion in an assay selected from: 1) a protectionassay involving direct challenge of previously vaccinated animals withclostridial neurotoxin; and 2) an assay of the neutralising capabilityof the anti-sera obtained in response to exposure of an animal to saidantigenic composition.
 29. An antigenic composition comprising a singlechain polypeptide comprising first and second domains, wherein: saidfirst domain is a clostridial neurotoxin light chain or a variantthereof, or a fragment of said light chain or variant wherein saidfragment or variant has a common antigenic cross-reactivity to saidclostridial neurotoxin light chain in an assay selected from: 1) aprotection assay involving direct challenge of previously vaccinatedanimals with clostridial neurotoxin; and 2) an assay of the neutralisingcapability of the anti-sera obtained in response to exposure of ananimal to said antigenic composition; and said second domain is aclostridial neurotoxin heavy chain H_(N) portion or a variant thereof,or a fragment of said heavy chain H_(N) portion or variant wherein saidvariant or fragment has a common antigenic cross-reactivity to saidclostridial neurotoxin heavy chain H_(N) portion; and wherein saidsecond domain is capable of (i) translocating the polypeptide into acell or (ii) increasing the solubility of the polypeptide compared tothe solubility of the first domain on its own or (iii) bothtranslocating the polypeptide into a cell and increasing the solubilityof the polypeptide compared to the solubility of the first domain on itsown; and wherein the second domain lacks a functional C-terminal part ofa clostridial neurotoxin heavy chain designated H_(C) thereby renderingthe polypeptide incapable of binding to cell surface receptors that arethe natural cell surface receptors to which native clostridialneurotoxin binds.
 30. An antigenic composition comprising a single chainpolypeptide comprising first and second domains, wherein: said firstdomain is a clostridial neurotoxin light chain or a variant thereof, ora fragment of said light chain or variant wherein said fragment orvariant has a common antigenic cross-reactivity to said clostridialneurotoxin light chain; and said second domain is a clostridialneurotoxin heavy chain H_(N) portion or a variant thereof, or a fragmentof said heavy chain H_(N) portion or variant wherein said variant orfragment has a common antigenic cross-reactivity to said clostridialneurotoxin heavy chain H_(N) portion in an assay selected from: 1) aprotection assay involving direct challenge of previously vaccinatedanimals with clostridial neurotoxin; and 2) an assay of the neutralisingcapability of the anti-sera obtained in response to exposure of ananimal to said antigenic composition; and wherein said second domain iscapable of (i) translocating the polypeptide into a cell or (ii)increasing the solubility of the polypeptide compared to the solubilityof the first domain on its own or (iii) both translocating thepolypeptide into a cell and increasing the solubility of the polypeptidecompared to the solubility of the first domain on its own; and whereinthe second domain lacks a functional C-terminal part of a clostridialneurotoxin heavy chain designated H_(C) thereby rendering thepolypeptide incapable of binding to cell surface receptors that are thenatural cell surface receptors to which native clostridial neurotoxinbinds.